System and method for the analysis of bodily fluids

ABSTRACT

A system for the rapid characterization of multi-analyte fluids, in one embodiment, includes a light source, a sensor array, and a detector. The sensor array is formed from a supporting member into which a plurality of cavities may be formed. A series of chemically sensitive particles are, in one embodiment positioned within the cavities. The particles may be configured to produce a signal when a receptor coupled to the particle interacts with the analyte. Using pattern recognition techniques, the analytes within a multi-analyte fluid may be characterized.

PRIORITY CLAIM

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/179,369 entitled “METHOD AND SYSTEM FOR COLLECTING ANDTRANSMITTING CHEMICAL INFORMATION,” filed Jan. 31, 2000, U.S.Provisional Application No. 60/179,424 entitled “SYSTEM AND METHOD FORTHE ANALYSIS OF BODILY FLUIDS” filed Jan. 31, 2000, U.S. ProvisionalApplication No. 60/179,294 entitled “SYSTEM AND METHOD FOR IDENTIFYINGNUCLEIC ACIDS IN A FLUID SAMPLE,” filed Jan. 31, 2000, U.S. ProvisionalApplication No. 60/179,380 entitled “METHOD OF PREPARING A SENSORARRAY,” filed Jan. 31, 2000, U.S. Provisional Application No. 60/179,292entitled “SYSTEM FOR TRANSFERRING FLUID SAMPLES THROUGH A SENSOR ARRAY,”filed Jan. 31, 2000 and U.S. Provisional Application No.60/179,293entitled “PORTABLE SENSOR ARRAY SYSTEM,” filed Jan. 31, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Research leading to this invention was federally supported, inpart, by grant No. 1R01GM57306-01 entitled “The Development of anElectronic Tongue” from the National Institute of Health and the U.S.Government has certain rights to this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to a method and device for thedetection of analytes in a fluid. More particularly, the inventionrelates to the development of a sensor array system capable ofdiscriminating mixtures of analytes, toxins, and/or bacteria in medical,food/beverage, and environmental solutions.

[0005] 2. Brief Description of the Related Art

[0006] The development of smart sensors capable of discriminatingdifferent analytes, toxins, and bacteria has become increasinglyimportant for clinical, environmental, health and safety, remotesensing, military, food/beverage and chemical processing applications.Although many sensors capable of high sensitivity and high selectivitydetection have been fashioned for single analyte detection, only in afew selected cases have array sensors been prepared which displaysolution phase multi-analyte detection capabilities. The advantages ofsuch array systems are their utility for the analysis of multipleanalytes and their ability to be “trained” to respond to new stimuli.Such on site adaptive analysis capabilities afforded by the arraystructures make their utilization promising for a variety of futureapplications. Array based sensors displaying the capacity to sense andidentify complex vapors have been demonstrated recently using a numberof distinct transduction schemes. For example, functional sensors basedon Surface Acoustic Wave (SAW), tin oxide (SnO₂) sensors, conductiveorganic polymers, and carbon black/polymer composites have beenfashioned. The use of tin oxide sensors, for example, is described inU.S. Pat. No. 5,654,497 to Hoffheins et al. These sensors display thecapacity to identify and discriminate between a variety of organicvapors by virtue of small site-to-site differences in responsecharacteristics. Pattern recognition of the overall fingerprint responsefor the array serves as the basis for an olfaction-like detection of thevapor phase analyte species. Indeed, several commercial “electronicnoses” have been developed recently. Most of the well establishedsensing elements are based on SnO₂ arrays which have been derivatized soas to yield chemically distinct response properties. Arrays based on SAWcrystals yield extremely sensitive responses to vapor, however,engineering challenges have prevented the creation of large SAW arrayshaving multiple sensor sites. To our knowledge, the largest SAW devicereported to date possesses only 12 sensor elements. Additionally,limited chemical diversity and the lack of understanding of themolecular features of such systems makes their expansion into morecomplex analysis difficult.

[0007] Other structures have been developed that are capable ofidentifying and discriminating volatile organic molecules. One structureinvolves a series of conductive polymer layers deposited onto metalcontacting layers. When these sensors are exposed to volatile reagents,some of the volatile reagents adsorb into the polymer layers, leading tosmall changes in the electrical resistance of these layers. It is thesmall differences in the behavior of the various sites that allows for adiscrimination, identification, and quantification of the vapors. Thedetection process takes only a few seconds, and sensitivities ofpart-per-billion can be achieved with this relatively simple approach.This “electronic nose” system is described in U.S. Pat. No. 5,698,089 toLewis et al. which is incorporated herein by reference as if set forthherein.

[0008] Although the above described electronic nose provides animpressive capability for monitoring volatile reagents, the systempossesses a number of undesirable characteristics that warrant thedevelopment of alternative sensor array systems. For example, theelectronic nose can be used only for the identification of volatilereagents. For many environmental, military, medical, and commercialapplications, the identification and quantification of analytes presentin liquid or solid-phase samples is necessary. Moreover, the electronicnose systems are expensive (e.g., the Aromascan system costs about$50,000/unit) and bulky (≧1 ft³). Furthermore, the functional elementsfor the currently available electronic nose are composed of conductivepolymer systems which possess little chemical selectivity for many ofthe analytes which are of interest to the military and civiliancommunities.

[0009] One of the most commonly employed sensing techniques hasexploited colloidal polymer microspheres for latex agglutination tests(LATs) in clinical analysis. Commercially available LATs for more than60 analytes are used routinely for the detection of infectious diseases,illegal drugs, and early pregnancy tests. The vast majority of thesetypes of sensors operate on the principle of agglutination of latexparticles (polymer microspheres) which occurs when theantibody-derivatized microspheres become effectively “cross-linked” by aforeign antigen resulting in the attachment to, or the inability to passthrough a filter. The dye-doped microspheres are then detectedcalorimetrically upon removal of the antigen carrying solution. However,the LATs lack the ability to be utilized for multiple, real time analytedetection schemes as the nature of the response intrinsically depends ona cooperative effect of the entire collection of microspheres.

[0010] Similar to the electronic nose, array sensors that have showngreat analytical promise are those based on the “DNA on a chip”technology. These devices possess a high density of DNA hybridizationsites that are affixed in a two-dimensional pattern on a planarsubstrate. To generate nucleotide sequence information, a pattern iscreated from unknown DNA fragments binding to various hybridizationsites. Both radiochemical and optical methods have provided excellentdetection limits for analysis of limited quantities of DNA. (Stimpson,D. I.; Hoijer, J. V.; Hsieh, W.; Jou, C.; Gardon, J.; Theriault, T.;Gamble, R.; Baldeschwieler, J. D. Proc. Natl. Acad. Sci. USA 1995, 92,6379). Although quite promising for the detection of DNA fragments,these arrays are generally not designed for non-DNA molecules, andaccordingly show very little sensitivity to smaller organic molecules.Many of the target molecules of interest to civilian and militarycommunities, however, do not possess DNA components. Thus, the need fora flexible, non-DNA based sensor is still desired. Moreover, while anumber of prototype DNA chips containing up to a few thousand differentnucleic acid probes have been described, the existing technologies tendto be difficult to expand to a practical size. As a result, DNA chipsmay be prohibitively expensive for practical uses.

[0011] Systems for analyzing fluid samples using an array formed ofheterogeneous, semi-selective thin films which function as sensingreceptor units are described in U.S. Pat. Nos. 6,023,540; 5,814,524;5,700,897; 5,512,490; 5,480,723; 5,252,494; 5,250,264; 5,244,813;5,244,636; and 5,143,853 which are incorporated herein by reference asif set forth herein. These systems appears to describe the use ofcovalently attached polymeric “cones” which are grown viaphotopolymerization onto the distal face of fiber optic bundles. Thesesensor probes appear to be designed with the goal of obtaining unique,continuous, and reproducible responses from small localized regions ofdye-doped polymer. The polymer appears to serve as a solid support forindicator molecules that provide information about test solutionsthrough changes in optical properties. These polymer supported sensorshave been used for the detection of analytes such as pH, metals, andspecific biological entities. Methods for manufacturing large numbers ofreproducible sensors, however, has yet to be developed. Moreover, nomethods for acquisitions of data streams in a simultaneous manner arecommercially available with this system. Optical alignment issues mayalso be problematic for these systems.

[0012] A method of rapid sample analysis for use in the diagnosticmicrobiology field is also desirable. The techniques now used for rapidmicrobiology diagnostics detect either antigens or nucleic acids. Rapidantigen testing is based on the use of antibodies to recognize eitherthe single cell organism or the presence of infected cell material.Inherent to this approach is the need to obtain and characterize thebinding of the antibody to unique structures on the organism beingtested. Since the identification and isolation of the appropriateantibodies is time consuming, these techniques are limited to a singleagent per testing module and there is no opportunity to evaluate theamount of agent present.

[0013] Most antibody methods are relatively insensitive and require thepresence of 10⁵ to 10⁷ organisms. The response time of antibody-antigenreactions in diagnostic tests of this type ranges from 10 to 120minutes, depending on the method of detection. The fastest methods aregenerally agglutination reactions, but these methods are less sensitivedue to difficulties in visual interpretation of the reactions.Approaches with slower reaction times include antigen recognition byantibody conjugated to either an enzyme or chromophore. These test typestend to be more sensitive, especially when spectrophotometric methodsare used to determine if an antigen-antibody reaction has occurred.These detection schemes do not, however, appear to allow thesimultaneous detection of multiple analytes on a single detectorplatform.

[0014] The alternative to antigen detection is the detection of nucleicacids. An approach for diagnostic testing with nucleic acids useshybridization to target unique regions of the target organism. Thesetechniques require fewer organisms (10³ to 10⁵), but require about fivehours to complete. As with antibody-antigen reactions this approach hasnot been developed for the simultaneous detection of multiple analytes.

[0015] The most recent improvement in the detection of microorganismshas been the use of nucleic acid amplification. Nucleic acidamplification tests have been developed that generate both qualitativeand quantitative data. However, the current limitations of these testingmethods are related to delays caused by specimen preparation,amplification, and detection. Currently, the standard assays requireabout five hours to complete. The ability to complete much fasterdetection for a variety of microorganisms would be of tremendousimportance to military intelligence, national safety, medical,environmental, and food areas.

[0016] It is therefore desirable that new sensors capable ofdiscriminating different analytes, toxins, and bacteria be developed formedical/clinical diagnostic, environmental, health and safety, remotesensing, military, food/beverage, and chemical processing applications.It is further desired that the sensing system be adaptable to thesimultaneous detection of a variety of analytes to improve throughputduring various chemical and biological analytical procedures.

SUMMARY OF THE INVENTION

[0017] Herein we describe a system and method for the analysis of afluid containing one or more analytes. The system may be used for eitherliquid or gaseous fluids. The system, in some embodiments, may generatepatterns that are diagnostic for both the individual analytes andmixtures of the analytes. The system in some embodiments, is made of aplurality of chemically sensitive particles, formed in an ordered array,capable of simultaneously detecting many different kinds of analytesrapidly. An aspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

[0018] In an embodiment of a system for detecting analytes, the system,in some embodiments, includes a light source, a sensor array, and adetector. The sensor array, in some embodiments, is formed of asupporting member which is configured to hold a variety of chemicallysensitive particles (herein referred to as “particles”) in an orderedarray. The particles are, in some embodiments, elements which willcreate a detectable signal in the presence of an analyte. The particlesmay produce optical (e.g., absorbance or reflectance) orfluorescence/phosphorescent signals upon exposure to an analyte.Examples of particles include, but are not limited to functionalizedpolymeric beads, agarous beads, dextrose beads, polyacrylamide beads,control

[0019] As depicted in FIG. 28C, additional layers of photoresistmaterial 766 and 768 may be formed upon the second photoresist layer764. The openings of the additional photoresist layers 766 and 768 maybe progressively larger as each layer is added to the stack. In thismanner, a tapered cavity may be formed. Additional layers of photoresistmaterial may be added until the desired thickness of the supportingmember is obtained. The thickness of the supporting member, in oneembodiment, is greater than a width of a particle. For example, if alayer of photoresist material has a thickness of about 25 μm and aparticle has a width of about 100 μm, a supporting member may be formedfrom four or more layers of photoresist material. While depicted aspyramidal, the cavity may be formed in a number of different shapes,including but not limited to, rectangular, circular, oval, triangular,and trapezoidal. Any of these shapes may be obtained by appropriatepatterning and etching of the photoresist layers as they are formed.

[0020] In some instances, the photoresist material may be substantiallytransparent to the light produced by the light source. As describedabove, the use of a transparent supporting member may lead to“cross-talk” between the cavities. To reduce the occurrence of this“cross-talk”, a substantially reflective layer 770 may be formed alongthe inner surface of the cavities 762, as depicted in FIG. 28D. In oneembodiment, the reflective layer is composed of a metal layer which isformed on the inner surface of the cavities 762. The metal layer may bedeposited using chemical vapor deposition or other techniques fordepositing thin metal layers. The presence of a reflective layer mayinhibit “cross-talk” between the cavities.

[0021] After the cavities 762 are formed, particles 772 may be insertedinto the cavities 762, as depicted in FIG. 28D. The narrow portions ofthe cavities 762 may serve as a support for the particles 772. Theparticles 772 may be inhibited from being displaced from the cavities762 by the lower portion of the cavities. After the particles 772 areplaced in the cavities 762, a cover 774 may be placed upon the uppersurface of the top layer 776 of the supporting member, as depicted inFIG. 28E. In one embodiment, the cover 774 is also formed from a film ofphotoresist material. After the cover layer is formed, openings 778 maybe formed in the cover 774 to allow the passage of the fluid into thecavities.

[0022] A high sensitivity CCD array may be used to measure changes inoptical characteristics which occur upon binding of thebiological/chemical agents. The CCD arrays may be interfaced withfilters, light sources, fluid delivery and micromachined particlereceptacles, so as to create a functional sensor array. Data acquisitionand handling may be performed with existing CCD technology. CCDdetectors may be configured to measure white light, ultraviolet light orfluorescence. Other detectors such as photomultiplier tubes, chargeinduction devices, photo diodes, photodiode arrays, and microchannelplates may also be used.

[0023] A particle, in some embodiments, possess both the ability to bindthe analyte of interest and to create a modulated signal. The particlemay include receptor molecules which posses the ability to bind theanalyte of interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte o f interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods. Some examples of naturalreceptors include, but are not limited to, DNA, RNA, proteins, enzymes,oligopeptides, antigens, and antibodies. Either natural or syntheticreceptors may be chosen for their ability to bind to the analytemolecules in a specific manner.

[0024] In one embodiment, a naturally occurring or synthetic receptor isbound to a polymeric bead in order to create the particle. The particle,in some embodiments, is capable of both binding the analyte(s) ofinterest and creating a detectable signal. In some embodiments, theparticle will create an optical signal when bound to an analyte ofinterest.

[0025] A variety of natural and synthetic receptors may be used. Thesynthetic receptors may come from a variety of classes including, butnot limited to, polynucleotides (e.g., aptamers), peptides (e.g.,enzymes and antibodies), synthetic receptors, polymeric unnaturalbiopolymers (e.g., polythioureas, polyguanidiniums), and imprintedpolymers. Polynucleotides are relatively small fragments of DNA whichmay be derived by sequentially building the DNA sequence. Peptidesinclude natural peptides such as antibodies or enzymes or may besynthesized from amino acids. Unnatural biopolymers are chemicalstructure which are based on natural biopolymers, but which are builtfrom unnatural linking units. For example, polythioureas andpolyguanidiniums have a structure similar to peptides, but may besynthesized from diamines (i.e., compounds which include at least twoamine functional groups) rather than amino acids. Synthetic receptorsare designed organic or inorganic structures capable of binding variousanalytes.

[0026] In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensed readilyby the described array sensors. Bacteria may also be detected using asimilar system. To detect, sense, and identify intact bacteria, the cellsurface of one bacteria may be differentiated from other bacteria, orgenomic material may be detected using oligonucleic receptors. Onemethod of accomplishing this differentiation is to target cell surfaceoligosaccharides (i.e., sugar residues). The use of synthetic receptorswhich are specific for oligosaccharides may be used to determine thepresence of specific bacteria by analyzing for cell surfaceoligosaccharides.

[0027] In one embodiment, a receptor may be coupled to a polymericresin. The receptor may undergo a chemical reaction in the presence ofan analyte such that a signal is produced. Indicators may be coupled tothe receptor or the polymeric bead. The chemical reaction of the analytewith the receptor may cause a change in the local microenvironment ofthe indicator to alter the spectroscopic properties of the indicator.This signal may be produced using a variety of signalling protocols.Such protocols may include absorbance, fluorescence resonance energytransfer, and/or fluorescence quenching. Receptor-analyte combinationmay include, but are not limited to, peptides-proteases,polynucleotides-nucleases, and oligosaccharides- oligosaccharidecleaving agents.

[0028] In one embodiment, a receptor and an indicator may be coupled toa polymeric resin. The receptor may undergo a conformational change inthe presence of an analyte such that a change in the localmicroenvironment of the indicator occurs. This change may alter thespectroscopic properties of the indicator. The interaction of thereceptor with the indicator may be produce a variety of differentsignals depending on the signalling protocol used. Such protocols mayinclude absorbance, fluorescence resonance energy transfer, and/orfluorescence quenching.

[0029] In an embodiment, the sensor array system includes an array ofparticles. The particles may include a receptor molecule coupled to apolymeric bead. The receptors, in some embodiments, are chosen forinteracting with analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles,while allowing the passage of the appropriate wavelengths of light. Thesupporting member may include a plurality of cavities. The cavities maybe formed such that at least one particle is substantially containedwithin the cavity. A vacuum may be coupled to the cavities. The vacuummay be applied to the entire sensor array. Alternatively, a vacuumapparatus may be coupled to the cavities to provide a vacuum to thecavities. A vacuum apparatus is any device capable of creating apressure differential to cause fluid movement. The vacuum apparatus mayapply a pulling force to any fluids within the cavity. The vacuumapparatus may pull the fluid through the cavity. Examples of vacuumapparatuss include pre-sealed vacuum chamber, vacuum pumps, vacuumlines, or aspirator-type pumps.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above brief description as well as further objects, featuresand advantages of the methods and apparatus of the present inventionwill be more fully appreciated by reference to the following detaileddescription of presently preferred but nonetheless illustrativeembodiments in accordance with the present invention when taken inconjunction with the accompanying drawings in which:

[0031]FIG. 1 depicts a schematic of an analyte detection system;

[0032]FIG. 2 depicts a particle disposed in a cavity;

[0033]FIG. 3 depicts a sensor array;

[0034] FIGS. 4A-F depict the formation of a Fabry-Perot cavity on theback of a sensor array;

[0035]FIG. 5 depicts the chemical constituents of a particle;

[0036]FIG. 6 depicts the chemical formulas of some receptor compounds;

[0037]FIG. 7 depicts a plot of the absorbance of green light vs.concentration of calcium (Ca⁺²) for a particle which includes ano-cresolphthalein complexone receptor;

[0038]FIG. 8 depicts a schematic view of the transfer of energy from afirst indicator to a second indicator in the presence of an analyte;

[0039]FIG. 9 depicts a schematic of the interaction of a sugar moleculewith a boronic acid based receptor.

[0040]FIG. 10 depicts various synthetic receptors;

[0041]FIG. 11 depicts a synthetic pathway for the synthesis ofpolythioureas;

[0042]FIG. 12 depicts a synthetic pathway for the synthesis ofpolyguanidiniums;

[0043]FIG. 13 depicts a synthetic pathway for the synthesis of diaminesfrom amino acids;

[0044]FIG. 14 depicts fluorescent diamino monomers;

[0045]FIG. 15 depicts a plot of counts/sec. (i.e., intensity) vs. timeas the pH of a solution surrounding a particle coupled too-cresolphthalein is cycled from acidic to basic conditions;

[0046]FIG. 16 depicts the color responses of a variety of sensingparticles to solutions of Ca⁺² and various pH levels;

[0047]FIG. 17 depicts an analyte detection system which includes asensor array disposed within a chamber;

[0048]FIG. 18 depicts an integrated analyte detection system;

[0049]FIG. 19 depicts a cross-sectional view of a cavity covered by amesh cover;

[0050]FIG. 20 depicts a top view of a cavity covered by a mesh cover;

[0051] FIGS. 21A-G depict a cross-sectional view of a series ofprocessing steps for the formation of a sensor array which includes aremovable top and bottom cover;

[0052] FIGS. 22A-G depict a cross-sectional view of a series ofprocessing steps for the formation of a sensor array which includes aremovable top and a stationary bottom cover;

[0053] FIGS. 23A-G depict a cross-sectional view of a series ofprocessing steps for the formation of a sensor array which includes aremovable top;

[0054] FIGS. 24A-D depict a cross-sectional view of a series ofprocessing steps for the formation of a silicon based sensor array whichincludes a top and bottom cover with openings aligned with the cavity;

[0055] FIGS. 25A-D depict a cross-sectional view of a series ofprocessing steps for the formation of a photoresist based sensor arraywhich includes a top and bottom cover with openings aligned with thecavity;

[0056] FIGS. 26A-E depict a cross-sectional view of a series ofprocessing steps for the formation of a plastic based sensor array whichincludes a top and bottom cover with openings aligned with the cavity;

[0057] FIGS. 27A-D depict a cross-sectional view of a series ofprocessing steps for the formation of a silicon based sensor array whichincludes a top cover with openings aligned with the cavity and a taperedcavity;

[0058] FIGS. 28A-E depict a cross-sectional view of a series ofprocessing steps for the formation of a photoresist based sensor arraywhich includes a top cover with openings aligned with the cavity and atapered cavity;

[0059] FIGS. 29A-E depict a cross-sectional view of a series ofprocessing steps for the formation of a photoresist based sensor arraywhich includes a top cover with openings aligned with the cavity and abottom cover;

[0060] FIGS. 30A-D depict a cross-sectional view of a series ofprocessing steps for the formation of a plastic based sensor array whichincludes a top cover with openings aligned with the cavity and a bottomcover;

[0061]FIG. 31 depicts a cross-sectional view of a schematic of amicropump;

[0062]FIG. 32 depicts a top view of an electrohydrodynamic pump;

[0063]FIG. 33 depicts a cross-sectional view of a sensor array whichincludes a micropump;

[0064]FIG. 34 depicts a cross-sectional view of a sensor array whichincludes a micropump and channels which are coupled to the cavities;

[0065]FIG. 35 depicts a cross-sectional view of a sensor array whichincludes multiple micropumps each micropump being coupled to a cavity;

[0066]FIG. 36 depicts a top view of a sensor array which includesmultiple electrohydrodynamic pumps;

[0067]FIG. 37 depicts a cross-sectional view of a sensor array whichincludes a system for delivering a reagent from a reagent particle to asensing cavity;

[0068]FIG. 38 depicts a cross-sectional view of a sensor array whichincludes a vacuum chamber;

[0069]FIG. 39 depicts a cross-sectional view of a sensor array whichincludes a vacuum chamber, a filter, and a reagent reservoir.

[0070]FIG. 40 depicts a general scheme for the testing of an antibodyanalyte;

[0071]FIG. 41 depicts general scheme for the detection of antibodieswhich uses a sensor array composed of four individual beads;

[0072]FIG. 42 depicts a sensor array which includes a vacuum chamber, asensor array chamber, and a sampling device;

[0073]FIG. 43 depicts a flow path of a fluid stream through a sensorarray from the top toward the bottom of the sensor array;

[0074]FIG. 44 depicts a flow path of a fluid stream through a sensorarray from the bottom toward the top of the sensor array;

[0075] FIGS. 45A-C depict the disruption of neuromuscular communicationby a toxin;

[0076]FIG. 45D depicts the attachment of differentially protected lysineto a bead;

[0077]FIG. 46 depicts a system for measuring the absorbance or emissionof a sensing particle;

[0078]FIG. 47 depicts receptors 3-6;

[0079]FIG. 48 depicts pH indicators which may be coupled to a particle;

[0080]FIG. 49 depicts a device for the analysis of IP₃ in cells;

[0081]FIG. 50 depicts the structure of Indo-1 and compound 2 and theemission spectra of Indo-1 and compound 2 in the presence of Ca(II) andCe(III), respectively;

[0082]FIG. 51 depicts a scheme wherein binding of citrate to a receptorfrees up the Indo-1 for Ca(II) binding;

[0083]FIG. 52 depicts the change in FRET between coumarin and5-carboxyfluorescein on resin beads as a function of the solvent;

[0084]FIG. 53 depicts a scheme wherein a signal of apo-7 to citrate istriggered by Cu(II) binding;

[0085]FIG. 54 depicts the response of receptor 3 and5-carboxyfluoroscein on a resin bead to the addition of citrate;

[0086] FIGS. 55A-I depict various sensing protocols forreceptor-indicator-polymeric resin particles;

[0087]FIG. 56 depicts a peptide trimer receptor and a pair offluorescent indicators coupled to a polymeric resin;

[0088]FIG. 57 depicts a synthetic scheme for anchoring dansyl anddapoxyl indicators to 6% agarose glyoxalated resin beads;

[0089]FIG. 58 depicts the RGB epifluorescence of 6 in EtOH with varyingratio buffer concentrations;

[0090]FIG. 59 depicts indicators and polymeric beads used forfluorescence studies;

[0091]FIG. 60 depicts Emission spectra of derivatized dapoxyl dyes invarious solvents;

[0092]FIG. 61 depicts a general structure of a chemically sensitiveparticle that includes a receptor and multiple indicators coupled to apolymeric resin;

[0093] FIGS. 62A-D depict various sensing protocols forreceptor-indicator-polymeric resin particles in which a cleavagereaction occurs;

[0094]FIG. 63 depicts a plot of the fluorescence signal of a chemicallysensitive particle in the presence of trypsin;

[0095]FIG. 64 depicts a block diagram illustrating a system forcollecting and transmitting chemical information over a computernetwork;

[0096]FIG. 65 depicts a flowchart of a method for collecting andtransmitting chemical information over a computer network;

[0097]FIG. 66 depicts a block diagram illustrating a system forcollecting and transmitting chemical information over a computernetwork;

[0098]FIG. 67 depicts a flowchart of a method for collecting andtransmitting chemical information over a computer network;

[0099]FIG. 68 depicts a block diagram illustrating a system forcollecting and transmitting chemical information over a computernetwork;

[0100]FIG. 69 depicts a flowchart of a method for collecting andtransmitting chemical information over a computer network;

[0101] FIGS. 70A-B depict a method of inserting particles into a sensorarray using a vacuum pickup dispenser head;

[0102] FIGS. 71A-B depict a method of inserting particles into a sensorarray using a solid dispenser head;

[0103] FIGS. 72A-D depict a method of inserting particles into a sensorarray using a vacuum chuck;

[0104]FIG. 73 depicts a cross section view of a sensor array whichincludes a passive pump system;

[0105]FIG. 74A depicts a top view of the sensor array of FIG. 57;

[0106]FIG. 74B depicts a bottom view of the sensor array of FIG. 57;

[0107] FIGS. 75A-D depict top views of the individual layers used toform a sensor array;

[0108]FIG. 76 depicts a top view of a sensor array which includesmultiple suites of arrays;

[0109]FIG. 77 depicts an alternate cross sectional view of a sensorarray which includes a passive transport system;

[0110]FIG. 78 depicts a portable sensor array system;

[0111]FIG. 79A-B depict views of an alternate portable sensor array;

[0112]FIG. 80 depicts an exploded view of a cartridge for use in aportable sensor array;

[0113]FIG. 81 depicts a cross sectional view of a cartridge for use in aportable sensor array; and

[0114]FIG. 82 depicts the placement of a particle into a cavity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0115] Herein we describe a system and method for the simultaneousanalysis of a fluid containing multiple analytes. The system may be usedfor either liquid or gaseous fluids. The system may generate patternsthat are diagnostic for both individual analytes and mixtures of theanalytes. The system, in some embodiments, is made of a combination ofchemically sensitive particles, formed in an ordered array, capable ofsimultaneously detecting many different kinds of analytes rapidly. Anaspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

[0116] System for Analysis of Analytes

[0117] Shown in FIG. 1 is an embodiment of a system for detectinganalytes in a fluid. The system, in some embodiments, includes a lightsource 110, a sensor array 120 and a detector 130. The light source 110may be a white light source or light emitting diodes (LED). In oneembodiment, light source 110 may be a blue light emitting diode (LED)for use in systems relying on changes in fluorescence signals. Forcalorimetric (e.g., absorbance) based systems, a white light source maybe used. The sensor array 120, in some embodiments, is formed of asupporting member which is configured to hold a variety of particles124. A detecting device 130 (e.g., a charge-coupled device “CCD”) may bepositioned below the sensor array to allow for data acquisition. Inanother embodiment, the detecting device 130 may be positioned above thesensor array.

[0118] Light originating from the light source 110, in some embodiments,passes through the sensor array 120 and out through the bottom side ofthe sensor array. The supporting member and the particles together, insome embodiments, provide an assembly whose optical properties are wellmatched for spectral analyses. Thus, light modulated by the particlesmay pass through the sensor array and onto the proximally spaceddetector 130. Evaluation of the optical changes may be completed byvisual inspection (e.g., with a microscope) or by use of amicroprocessor 140 coupled to the detector. For fluorescencemeasurements, a filter 135 may be placed between supporting member 120and detector 130 to remove the excitation wavelength. A fluid deliverysystem 160 may be coupled to the supporting member. The fluid deliverysystem 160 may be configured to introduce samples into and out of thesensor array.

[0119] In an embodiment, the sensor array system includes an array ofparticles. Upon the surface and within the interior region of theparticles are, in some embodiments, located a variety of receptors forinteracting with analytes. The supporting member, in some embodiments,is used to localize these particles as well as to serve as amicroenvironment in which the chemical assays can be performed. For thechemical/biological agent sensor arrays, the particles used for analysisare about 0.05-500 microns in diameter, and may actually change size(e.g., swell or shrink) when the chemical environment changes.Typically, these changes occur when the array system is exposed to thefluid stream which includes the analytes. For example, a fluid streamwhich comprises a non-polar solvent, may cause non-polar particles tochange in volume when the particles are exposed to the solvent. Toaccommodate these changes, it is preferred that the supporting memberconsist of an array of cavities which serve as micro test-tubes.

[0120] The supporting member may be made of any material capable ofsupporting the particles, while allowing the passage of the appropriatewavelength of light. The supporting member is also made of a materialsubstantially impervious to the fluid in which the analyte is present. Avariety of materials may be used including plastics, glass, siliconbased materials (e.g., silicon, silicon dioxide, silicon nitride, etc.)and metals. In one embodiment, the supporting member includes aplurality of cavities. The cavities may be formed such that at least oneparticle is substantially contained within the cavity. Alternatively, aplurality of particles may be contained within a single cavity.

[0121] In an embodiment, the supporting member may consist of a strip ofplastic which is substantially transparent to the wavelength of lightnecessary for detection. A series of cavities may be formed within thestrip. The cavities may be configured to hold at least one particle. Theparticles may be contained within the strip by a transparent cover whichis configured to allow passage of the analyte containing fluid into thecavities.

[0122] In another embodiment, the supporting member may be formed usinga silicon wafer as depicted in FIG. 2. The silicon wafer 210 may includea substantially transparent layer 220 formed on the bottom surface ofthe wafer. The cavities 230, in one embodiment, are formed by ananisotropic etch process of the silicon wafer. In one embodiment,anisotropic etching of the silicon wafer is accomplished using a wethydroxide etch. Photolithographic techniques may be used to define thelocations of the cavities. The cavities may be formed such that thesidewalls of the cavities are substantially tapered at an angle ofbetween about 50 to 60 degrees. Formation of such angled cavities may beaccomplished by wet anisotropic etching of <100> silicon. The term“<100> silicon” refers to the crystal orientation of the silicon wafer.Other types of silicon, (e.g., <110> and <111> silicon) may lead tosteeper angled sidewalls. For example, <111> silicon may lead tosidewalls formed at about 90 degrees. The angled sides of the cavitiesin some embodiments, serve as “mirror layers” which may improve thelight collection efficiency of the cavities. The etch process may becontrolled so that the formed cavities extend through the silicon waferto the upper surface of transparent layer 220. While depicted aspyramidal, the cavities may be formed in a number of shapes includingbut not limited to, spherical, oval, cubic, or rectangular. An advantageto using a silicon wafer for the support member, is that the siliconmaterial is substantially opaque to the light produced from the lightsource. Thus, the light may be inhibited from passing from one cavity toadjacent cavities. In this manner, light from one cavity may beinhibited from influencing the spectroscopic changes produced in anadjacent cavity.

[0123] The silicon wafer, in some embodiments, has an area ofapproximately 1 cm to about 100 cm² and includes about 10¹ to about 10⁶cavities. In an embodiment, about 100 cavities are formed in a ten byten matrix. The center to center distance between the cavities, in someembodiments, is about 500 microns. Each of the cavities may include atleast one particle.

[0124] The transparent layer 220 may serve as a window, allowing lightof a variety of wavelengths to pass through the cavities 230 and to thedetector. Additionally, the transparent layer may serve as a platformonto which the individual particles 235 may be positioned. Thetransparent layer may be formed of silicon dioxide (SiO₂), siliconnitride (Si₃N₄) or silicon dioxide/silicon nitride multi-layer stacks.The transparent layer, in some embodiments, is deposited onto thesilicon wafer prior to the formation of the cavities.

[0125] The cavities 230 may be sized to substantially contain a particle235. The cavities are, in some embodiments, larger than a particle. Thecavities are, in some embodiments, sized to allow facile placement andremoval of the particle within the cavities. The cavity may besubstantially larger than the particle, thus allowing the particle toswell during use. For example, a particle may have a size as depicted inFIG. 2 by particle 235. During use, contact with a fluid (e.g., asolvent) may cause the particle to swell, for example, to a sizedepicted as circle 236. In some embodiments, the cavity is sized toallow such swelling of the particle during use. A particle may bepositioned at the bottom of a cavity using, e.g., a micromanipulator.After a particle has been placed within the cavity, a transparent coverplate 240 may be placed on top of the supporting member to keep theparticle in place.

[0126] When forming an array which includes a plurality of particles,the particles may be placed in the array in an ordered fashion using themicromanipulator. In this manner, a ordered array having a predefinedconfiguration of particles may be formed. Alternatively, the particlesmay be randomly placed within the cavities. The array may subsequentlyundergo a calibration test to determine the identity of the particle atany specified location in the supporting member.

[0127] The transparent cover plate 240, in some embodiments, is coupledto the upper surface of the silicon wafer 220 such that the particlesare inhibited from becoming dislodged from the cavity. The transparentcover plate, in some embodiments, is positioned a fixed distance abovethe silicon wafer, as depicted in FIG. 2, to keep the particle in place,while allowing the entrance of fluids into the cavities. The transparentcover plate, in some embodiments, is positioned at a distance above thesubstrate which is substantially less than a width of the particle. Thetransparent cover plate may be made of any material which issubstantially transparent to the wavelength of light being utilized bythe detector. The transparent cover plate may be made of plastic, glass,quartz, or silicon dioxide/silicon nitride.

[0128] In one embodiment, the transparent cover plate 240, is a thinsheet of glass (e.g., a microscope slide cover slip). The slide may bepositioned a fixed distance above the silicon wafer. Support structures241 (See FIG. 2) may be placed upon the silicon wafer 210 to positionthe transparent cover plate 240. The support structures may be formedfrom a polymer or a silicon based material. In another embodiment, apolymeric substrate is coupled to the silicon wafer to form the supportstructures 241 for the transparent cover plate 240. In an embodiment, aplastic material with an adhesive backing (e.g., cellophane tape) ispositioned on the silicon wafer 210. After the support structures 241are placed on the wafer the transparent cover plate 240 is placed uponthe support structures. The support structures inhibit the transparentcover sheet from contacting the silicon wafer 200. In this manner, achannel is formed between the silicon wafer and the transparent coverplate which allow the fluid to pass into the cavity, while inhibitingdisplacement of the particle by the fluid.

[0129] In another embodiment, the transparent cover plate 240 may befastened to the upper surface of the silicon wafer, as depicted in FIG.3. In this embodiment, the fluid may be inhibited from entering thecavities 230 by the transparent cover plate 240. To allow passage of thefluid into the cavities, a number of channels 250 may be formed in thesilicon wafer. The channels, in one embodiment, are oriented to allowpassage of the fluid into substantially all of the cavities. Whencontacted with the fluid, the particles may swell to a size which mayplug the channels. To prevent this plugging, the channels may be formednear the upper portion of the cavities, as depicted in FIG. 3. Thechannels, in one embodiment, are formed using standard photolithographicmasking to define the regions where the trenches are to be formed,followed by the use of standard etching techniques. A depth of thecavity may be such that the particle resides substantially below theposition of the channel. In this way, the plugging of the channels dueto swelling of the particle may be prevented.

[0130] The inner surfaces of the cavities may be coated with a materialto aid the positioning of the particles within the cavities. In oneembodiment, a thin layer of gold or silver may be used to line the innersurface of the cavities. The gold or silver layer may act as ananchoring surface to anchor particles (e.g., via alkylthiol bonding). Inaddition, the gold or silver layer may also increase the reflectivity ofthe inner surface of the cavities. The increased reflectance of thesurface may enhance the analyte detection sensitivity of the system.Alternatively, polymer layers and self-assembled monolayers formed uponthe inner surface of the cavities may be used to control the particleadhesion interactions. Additional chemical anchoring methods may be usedfor silicon surfaces such as those based on siloxane type reagents,which may be attached to Si—OH functionalities. Similarly, monomeric andpolymeric reagents attached to an interior region of the cavities can beused to alter the local wetting characteristics of the cavities. Thistype of methodology can be used to anchor the particles as well as toalter the fluid delivery characteristics of the cavity. Furthermore,amplification of the signals for the analytes may be accomplished withthis type of strategy by causing preconcentration of appropriateanalytes in the appropriate type of chemical environment.

[0131] In another embodiment, the optical detector may be integratedwithin the bottom transparent layer 220 of the supporting member, ratherthan using a separate detecting device. The optical detectors may beformed using a semiconductor-based photodetector 255. The opticaldetectors may be coupled to a microprocessor to allow evaluation offluids without the use of separate detecting components. Additionally,the fluid delivery system may also be incorporated into the supportingmember. Micro-pumps and micro-valves may also be incorporated into thesilicon wafer to aid passage of the fluid through the cavities.Integration of detectors and a fluid delivery system into the supportingmember may allow the formation of a compact and portable analyte sensingsystem. Optical filters may also be integrated into the bottom membraneof the cavities. These filters may prevent short wavelength excitationfrom producing “false” signals in the optical detection system (e.g., aCCD detector array) during fluorescence measurements.

[0132] A sensing cavity may be formed on the bottom surface of thesupport substrate. An example of a sensing cavity that may be used is aFabry-Perot type cavity. Fabry-Perot cavity-based sensors may be used todetect changes in optical path length induced by either a change in therefractive index or a change in physical length of the cavity. Usingmicromachining techniques, Fabry-Perot sensors may be formed on thebottom surface of the cavity.

[0133] FIGS. 4A-F depict a sequence of processing steps for theformation of a cavity and a planar top diaphragm Fabry-Perot sensor onthe bottom surface of a silicon based supporting member. A sacrificialbarrier layer 262 a/b is deposited upon both sides of a siliconsupporting member 260. The silicon supporting member 260 may be adouble-side polished silicon wafer having a thickness ranging from about100 μm to about 500 μm, preferably from about 200 μm to about 400 μm,and more preferably of about 300 μm. The barrier layer 262 a/b may becomposed of silicon dioxide, silicon nitride, or silicon oxynitride. Inone embodiment, the barrier layer 262 a/b is composed of a stack ofdielectric materials. As depicted in FIG. 4A, the barrier layer 262 a/bis composed of a stack of dielectric materials which includes a siliconnitride layer 271 a/b and a silicon dioxide layer 272 a/b. Both layersmay be deposited using a low pressure chemical vapor deposition(“LPCVD”) process. Silicon nitride may be deposited using an LPCVDreactor by reaction of ammonia (NH₃) and dichlorosilane (SiCl₂H₂) at agas flow rate of about 3.5:1, a temperature of about 800° C., and apressure of about 220 mTorr. The silicon nitride layer 271 a/b isdeposited to a thickness in the range from about 100 Å to about 500 Å,preferably from 200 Å to about 400 Å, and more preferably of about 300Å. Silicon dioxide is may be deposited using an LPCVD reactor byreaction of silane (SiH₄) and oxygen (O₂) at a gas flow rate of about3:4, a temperature of about 450° C., and a pressure of about 110 mTorr.The silicon dioxide layer 272 a/b is deposited to a thickness in therange from about 3000 Å to about 7000 Å, preferably from 4000 Å to about6000 Å, and more preferably of about 5000 Å. The front face silicondioxide layer 272 a, in one embodiment, acts as the main barrier layer.The underlying silicon nitride layer 271 a acts as an intermediatebarrier layer to inhibit overetching of the main barrier layer duringsubsequent KOH wet anisotropic etching steps.

[0134] A bottom diaphragm layer 264 a/b is deposited upon the barrierlayer 262 a/b on both sides of the supporting member 260. The bottomdiaphragm layer 264 a/b may be composed of silicon nitride, silicondioxide, or silicon oxynitride. In one embodiment, the bottom diaphragmlayer 264 a/b is composed of a stack of dielectric materials. Asdepicted in FIG. 4A, the bottom diaphragm layer 264 a/b is composed of astack of dielectric materials which includes a pair of silicon nitridelayers 273 a/b and 275 a/b surrounding a silicon dioxide layer 274 a/b.All of the layers may be deposited using an LPCVD process. The siliconnitride layers 273 a/b and 275 a/b have a thickness in the range fromabout 500 Å to about 1000 Å, preferably from 700 Å to about 800 Å, andmore preferably of about 750 Å. The silicon dioxide layer 274 a/b has athickness in the range from about 3000 Å to about 7000 Å, preferablyfrom 4000 Å to about 6000 Å, and more preferably of about 4500 Å.

[0135] A cavity which will hold the particle may now be formed in thesupporting member 260. The bottom diaphragm layer 264 b and the barrierlayer 262 b formed on the back side 261 of the silicon supporting member260 are patterned and etched using standard photolithographictechniques. In one embodiment, the layers are subjected to a plasma etchprocess. The plasma etching of silicon dioxide and silicon nitride maybe performed using a mixture of carbontetrafluoride (CF₄) and oxygen(O₂). The patterned back side layers 262 b and 264 b may be used as amask for anisotropic etching of the silicon supporting member 260. Thesilicon supporting member 260, in one embodiment, is anisotropicallyetched with a 40% potassium hydroxide (“KOH”) solution at 80° C. to formthe cavity. The etch is stopped when the front side silicon nitridelayer 271 a is reached, as depicted in FIG. 4B. The silicon nitridelayer 271 a inhibits etching of the main barrier layer 272 a during thisetch process. The cavity 267 may be formed extending through thesupporting member 260. After formation of the cavity, the remainingportions of the back side barrier layer 262 b and the diaphragm layer264 b may be removed.

[0136] Etch windows 266 are formed through the bottom diaphragm layer264 a on the front side of the wafer. A masking layer (not shown) isformed over the bottom diaphragm layer 264 a and patterned usingstandard photolithographic techniques. Using the masking layer, etchwindows 266 may be formed using a plasma etch. The plasma etching ofsilicon dioxide and silicon nitride may be performed using a mixture ofcarbontetrafluoride (CF₄) and oxygen (O₂). The etching is continuedthrough the bottom diaphragm layer 264 a and partially into the barrierlayer 262 a. In one embodiment, the etching is stopped at approximatelyhalf the thickness of the barrier layer 262 a. Thus, when the barrierlayer 262 a is subsequently removed the etch windows 266 will extendthrough the bottom diaphragm layer 264 a, communicating with the cavity267. By stopping the etching at a midpoint of the barrier layer, voidsor discontinuities may be reduced since the bottom diaphragm is stillcontinuous due to the remaining barrier layer.

[0137] After the etch windows 266 are formed, a sacrificial spacer layer268 a/b is deposited upon the bottom diaphragm layer 264 a and withincavity 267, as depicted in FIG. 4C. The spacer layer may be formed fromLPCVD polysilicon. In one embodiment, the front side deposited spacerlayer 268 a will also at least partially fill the etch windows 266.Polysilicon may be deposited using an LPCVD reactor using silane (SiH₄)at a temperature of about 650° C. The spacer layer 268 a/b is depositedto a thickness in the range from about 4000 Å to about 10,000 Å,preferably from 6000 Å to about 8000 Å, and more preferably of about7000 Å. The preferred thickness of the spacer layer 268 a is dependenton the desired thickness of the internal air cavity of the Fabry-Perotdetector. For example, if a Fabry-Perot detector which is to include a7000 Å air cavity between the top and bottom diaphragm layer is desired,a spacer layer having a thickness of about 7000 Å would be formed. Afterthe spacer layer has been deposited, a masking layer for etching thespacer layer 268 a (not shown) is used to define the etch regions of thespacer layer 268 a. The etching may be performed using a composition ofnitric acid (HNO₃), water, and hydrogen fluoride (HF) in a ratio of25:13:1, respectively, by volume. The lateral size of the subsequentlyformed cavity is determined by the masking pattern used to define theetch regions of the spacer layer 268 a.

[0138] After the spacer layer 268 a has been etched, the top diaphragmlayer 270 a/b is formed.

[0139] The top diaphragm 270 a/b, in one embodiment, is deposited uponthe spacer layer 268 a/b on both sides of the supporting member. The topdiaphragm 270 a/b may be composed of silicon nitride, silicon dioxide,or silicon oxynitride. In one embodiment, the top diaphragm 270 a/b iscomposed of a stack of dielectric materials. As depicted in FIG. 4C, thetop diaphragm 270 a/b is composed of a stack of dielectric materialswhich includes a pair of silicon nitride layers 283 a/b and 285 a/bsurrounding a silicon dioxide layer 284 a/b. All of the layers may bedeposited using an LPCVD process. The silicon nitride layers 283 a/b and285 a/b have a thickness in the range from about 1000 Å to about 2000 Å,preferably from 1200 Å to about 1700 Å, and more preferably of about1500 Å. The silicon dioxide layer 284 a/b has a thickness in the rangefrom about 5000 Å to about 15,500 Å, preferably from 7500 Å to about12,000 Å, and more preferably of about 10,500 Å.

[0140] After depositing the top diaphragm 270 a/b, all of the layersstacked on the bottom face of the supporting member (e.g., layers 268 b,283 b, 284 b, and 285 b) are removed by multiple wet and plasma etchingsteps, as depicted in FIG. 4D. After these layers are removed, the nowexposed portions of the barrier layer 262 a are also removed. Thisexposes the spacer layer 268 a which is present in the etch windows 266.The spacer layer 268 may be removed from between the top diaphragm 270 aand the bottom diaphragm 264 a by a wet etch using a KOH solution, asdepicted in FIG. 4D. Removal of the spacer material 268 a, forms acavity 286 between the top diaphragm layer 270 a and the bottomdiaphragm layer 264 a. After removal of the spacer material, the cavity286 may be washed using deionized water, followed by isopropyl alcoholto clean out any remaining etching solution.

[0141] The cavity 286 of the Fabry-Perot sensor may be filled with asensing substrate 290, as depicted in FIG. 4E. To coat the cavity 286with a sensing substrate 290, the sensing substrate may be dissolved ina solvent. A solution of the sensing substrate is applied to thesupporting member 260. The solution is believed to rapidly enter thecavity 286 through the etched windows 266 in the bottom diaphragm 264 a,aided in part by capillary action. As the solvent evaporates, a thinfilm of the sensing substrate 290 coats the inner walls of the cavity286, as well as the outer surface of the bottom diaphragm 264 a. Byrepeated treatment of the supporting member with the solution of thesensing substrate, the thickness of the sensing substrate may be varied.

[0142] In one embodiment, the sensing substrate 290 ispoly(3-dodecylthiophene) whose optical properties change in response tochanges in oxidation states. The sensing substratepoly(3-dodecylthiophene) may be dissolved in a solvent such aschloroform or xylene. In one embodiment, a concentration of about 0.1 gof poly(3-dodecylthiophene)/mL is used. Application of the solution ofpoly(3-dodecylthiophene) to the supporting member causes a thin film ofpoly(3-dodecylthiophene) to be formed on the inner surface of thecavity.

[0143] In some instances, the sensing substrate, when deposited within acavity of a Fabry-Perot type detector, may cause stress in the topdiaphragm of the detector. It is believed that when a sensing polymercoats a planar top diaphragm, extra residual stress on the top diaphragmcauses the diaphragm to become deflected toward the bottom diaphragm. Ifthe deflection becomes to severe, sticking between the top and bottomdiaphragms may occur. In one embodiment, this stress may be relieved bythe use of supporting members 292 formed within the cavity 286, asdepicted in FIG. 4F. The supporting members 292 may be formed withoutany extra processing steps to the above described process flow. Theformation of supporting members may be accomplished by deliberatelyleaving a portion of the spacer layer within the cavity. This may beaccomplished by underetching the spacer layer (e.g., terminating theetch process before the entire etch process is finished). The remainingspacer will behave as a support member to reduce the deflection of thetop diaphragm member. The size and shape of the support members may beadjusted by altering the etch time of the spacer layer, or adjusting theshape of the etch windows 266.

[0144] In another embodiment, a high sensitivity CCD array may be usedto measure changes in optical characteristics which occur upon bindingof the biological/chemical agents. The CCD arrays may be interfaced withfilters, light sources, fluid delivery and micromachined particlereceptacles, so as to create a functional sensor array. Data acquisitionand handling may be performed with existing CCD technology. Data streams(e.g., red, green, blue for calorimetric assays; gray intensity forfluorescence assays) may be transferred from the CCD to a computer via adata acquisition board. Current CCDs may allow for read-out rates of 10pixels per second. Thus, the entire array of particles may be evaluatedhundreds of times per second allowing for studies of the dynamics of thevarious host-guest interaction rates as well as the analyte/polymerdiffusional characteristics. Evaluation of this data may offer a methodof identifying and quantifying the chemical/biological composition ofthe test samples. CCD detectors may be configured to measure whitelight, ultraviolet light or fluorescence. Other detectors such asphotomultiplier tubes, charge induction devices, photodiode, photodiodearrays, and microchannel plates may also be used. It should beunderstood that while the detector is depicted as being positioned underthe supporting member, the detector may also be positioned above thesupporting member. It should also be understood that the detectortypically includes a sensing element for detecting the spectroscopicevents and a component for displaying the detected events. The displaycomponent may be physically separated from the sensing element. Thesensing element may be positioned above or below the sensor array whilethe display component is positioned close to a user.

[0145] In one embodiment, a CCD detector may be used to record colorchanges of the chemical sensitive particles during analysis. As depictedin FIG. 1, a CCD detector 130 may be placed beneath the supportingmember 120. The light transmitted through the cavities is captured andanalyzed by the CCD detector. In one embodiment, the light is brokendown into three color components, red, green and blue. To simplify thedata, each color is recorded using 8 bits of data. Thus, the data foreach of the colors will appear as a value between 0 and 255. The colorof each chemical sensitive element may be represented as a red, blue andgreen value. For example, a blank particle (i.e., a particle which doesnot include a receptor) will typically appear white. For example, whenbroken down into the red, green and blue components, it is found that atypical blank particle exhibits a red value of about 253, a green valueof about 250, and a blue value of about 222. This signifies that a blankparticle does not significantly absorb red, green or blue light. When aparticle with a receptor is scanned, the particle may exhibit a colorchange, due to absorbance by the receptor. For example, it was foundthat when a particle which includes a 5-carboxyfluorescein receptor issubjected to white light, the particle shows a strong absorbance of bluelight. The CCD detector values for the 5-carboxyfluorescein particleexhibits a red value of about 254, a green value of about 218, and ablue value of about 57. The decrease in transmittance of blue light isbelieved to be due to the absorbance of blue light by the5-carboxyfluorescein. In this manner, the color changes of a particlemay be quantitatively characterized. An advantage of using a CCDdetector to monitor the color changes is that color changes which maynot be noticeable to the human eye may now be detected.

[0146] The support array may be configured to allow a variety ofdetection modes to be practiced. In one embodiment, a light source isused to generate light which is directed toward the particles. Theparticles may absorb a portion of the light as the light illuminates theparticles. The light then reaches the detector, reduced in intensity bythe absorbance of the particles. The detector may be configure tomeasure the reduction in light intensity (i.e., the absorbance) due tothe particles. In another embodiment, the detector may be placed abovethe supporting member. The detector may be configured to measure theamount of light reflected off of the particles. The absorbance of lightby the particles is manifested by a reduction in the amount of lightbeing reflected from the cavity. The light source in either embodimentmay be a white light source or a fluorescent light source.

[0147] Chemically Sensitive Particles

[0148] A particle, in some embodiments, possess both the ability to bindthe analyte of interest and to create a modulated signal. The particlemay include receptor molecules which posses the ability to bind theanalyte of interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte o f interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods. Some examples of naturalreceptors include, but are not limited to, DNA, RNA, proteins, enzymes,oligopeptides, antigens, and antibodies. Either natural or syntheticreceptors may be chosen for their ability to bind to the analytemolecules in a specific manner. The forces which driveassociation/recognition between molecules include the hydrophobiceffect, anion-cation attraction, and hydrogen bonding. The relativestrengths of these forces depend upon factors such as the solventdielectric properties, the shape of the host molecule, and how itcomplements the guest. Upon host-guest association, attractiveinteractions occur and the molecules stick together. The most widelyused analogy for this chemical interaction is that of a “lock and key”.The fit of the key molecule (the guest) into the lock (the host) is amolecular recognition event.

[0149] A naturally occurring or synthetic receptor may be bound to apolymeric resin in order to create the particle. The polymeric resin maybe made from a variety of polymers including, but not limited to,agarous, dextrose, acrylamide, control pore glass beads,polystyrene-polyethylene glycol resin, polystyrene-divinyl benzeneresin, formylpolystyrene resin, trityl-polystyrene resin, acetylpolystyrene resin, chloroacetyl polystyrene resin, aminomethylpolystyrene-divinylbenzene resin, carboxypolystyrene resin,chloromethylated polystyrene-divinylbenzene resin, hydroxymethylpolystyrene-divinylbenzene resin, 2-chlorotrityl chloride polystyreneresin, 4-benzyloxy-2′4′-dimethoxybenzhydrol resin (Rink Acid resin),triphenyl methanol polystyrene resin, diphenylmethanol resin, benzhydrolresin, succinimidyl carbonate resin, p-nitrophenyl carbonate resin,imidazole carbonate resin, polyacrylamide resin,4-sulfamylbenzoyl-4′-methylbenzhydrylamine-resin (Safety-catch resin),2-amino-2-(2′-nitrophenyl) propionic acid-aminomethyl resin (ANP Resin),p-benzyloxybenzyl alcohol-divinylbenzene resin (Wang resin),p-methylbenzhydrylamine-divinylbenzene resin (MBHA resin),Fmoc-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine linked to resin(Knorr resin), 4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin(Rink resin), 4-hydroxymethyl-benzoyl-4′-methylbenzhydrylamine resin(HMBA-MBHA Resin), p-nitrobenzophenone oxime resin (Kaiser oxime resin),and amino-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine handlelinked to 2-chlorotrityl resin (Knorr-2-chlorotrityl resin). In oneembodiment, the material used to form the polymeric resin is compatiblewith the solvent in which the analyte is dissolved. For example,polystyrene-divinyl benzene resin will swell within non-polar solvents,but does not significantly swell within polar solvents. Thus,polystyrene-divinyl benzene resin may be used for the analysis ofanalytes within non-polar solvents. Alternatively,polystyrene-polyethylene glycol resin will swell with polar solventssuch as water. Polystyrene-polyethylene glycol resin may be useful forthe analysis of aqueous fluids.

[0150] In one embodiment, a polystyrene-polyethylene glycol-divinylbenzene material is used to form the polymeric resin. Thepolystyrene-polyethylene glycol-divinyl benzene resin is formed from amixture of polystyrene 375, divinyl benzene 380 andpolystyrene-polyethylene glycol 385, see FIG. 5. The polyethylene glycolportion of the polystyrene-polyethylene glycol 385, in one embodiment,may be terminated with an amine. The amine serves as a chemical handleto anchor both receptors and indicator dyes. Other chemical functionalgroups may be positioned at the terminal end of the polyethylene glycolto allow appropriate coupling of the polymeric resin to the receptormolecules or indicators.

[0151] The chemically sensitive particle, in one embodiment, is capableof both binding the analyte(s) of interest and creating a detectablesignal. In one embodiment, the particle will create an optical signalwhen bound to an analyte of interest. The use of such a polymeric boundreceptors offers advantages both in terms of cost and configurability.Instead of having to synthesize or attach a receptor directly to asupporting member, the polymeric bound receptors may be synthesized enmasse and distributed to multiple different supporting members. Thisallows the cost of the sensor array, a major hurdle to the developmentof mass-produced environmental probes and medical diagnostics, to bereduced. Additionally, sensor arrays which incorporate polymeric boundreceptors may be reconfigured much more quickly than array systems inwhich the receptor is attached directly tot he supporting member. Forexample, if a new variant of a pathogen or a pathogen that contains agenetically engineered protein is a threat, then a new sensor arraysystem may be readily created to detect these modified analytes bysimply adding new sensor elements (e.g., polymeric bound receptors) to apreviously formed supporting member.

[0152] In one embodiment, a receptor, which is sensitive to changes inthe pH of a fluid sample is bound to a polymeric resin to create aparticle. That is, the receptor is sensitive to the concentration ofhydrogen cations (H⁺). The receptor in this case is typically sensitiveto the concentration of ft in a fluid solution. The analyte of interestmay therefore be H⁺. There are many types of molecules which undergo acolor change when the pH of the fluid is changed. For example, manytypes of dyes undergo significant color changes as the pH of the fluidmedium is altered. Examples of receptors which may be used to monitorthe pH of a fluid sample include 5-carboxyfluorescein and alizarincomplexone, depicted in FIG. 6. Each of these receptors undergoessignificant color changes as the pH of the fluid is altered.5-carboxyfluorescein undergoes a change from yellow to orange as the pHof the fluid is increased. Alizarin complexone undergoes two colorchanges, first from yellow to red, then from red to blue as the pH ofthe fluid increases. By monitoring the change in color caused by dyesattached to a polymeric particle, the pH of a solution may bequalitatively and, with the use of a detector (e.g., a CCD detector),quantitatively monitored.

[0153] In another embodiment, a receptor which is sensitive to presenceof metal cations is bound to a polymeric particle to create a particle.The receptor in this case is typically sensitive to the concentration ofone or more metal cations present in a fluid solution. In general,colored molecules which will bind cations may be used to determine thepresence of a metal cation in a fluid solution. Examples of receptorswhich may be used to monitor the presence of cations in a fluid sampleinclude alizarin complexone and o-cresolphthalein complexone, see FIG.6. Each of these receptors undergoes significant color changes as theconcentration of a specific metal ion in the fluid is altered. Alizarincomplexone is particularly sensitive to lanthanum ions. In the absenceof lanthanum, alizarin complexone will exhibit a yellow color. As theconcentration of lanthanum is increased, alizarin complexone will changeto a red color. o-Cresolphthalein complexone is particularly sensitiveto calcium ions. In the absence of calcium, o-cresolphthalein complexoneis colorless. As the concentration of calcium is increased,o-cresolphthalein complexone will change to a blue color. By monitoringthe change in color of metal cation sensitive receptors attached to apolymeric particle, the presence of a specific metal ion may bequalitatively and, with the use of a detector (e.g., a CCD detector),quantitatively monitored.

[0154] Referring to FIG. 7, a graph of the absorbance of green light vs.concentration of calcium (Ca⁺²) is depicted for a particle whichincludes an o-cresolphthalein complexone receptor. As the concentrationof calcium is increased, the absorbance of green light increases in alinear manner up to a concentration of about 0.0006 M. A concentrationof 0.0006 M is the solubility limit of calcium in the fluid, thus nosignificant change in absorbance is noted after this point. The linearrelationship between concentration and absorbance allows theconcentration of calcium to be determined by measuring the absorbance ofthe fluid sample.

[0155] In one embodiment, a detectable signal may be caused by thealtering of the physical properties of an indicator ligand bound to thereceptor or the polymeric resin. In one embodiment, two differentindicators are attached to a receptor or the polymeric resin. When ananalyte is captured by the receptor, the physical distance between thetwo indicators may be altered such that a change in the spectroscopicproperties of the indicators is produced. A variety of fluorescent andphosphorescent indicators may be used for this sensing scheme. Thisprocess, known as Forster energy transfer, is extremely sensitive tosmall changes in the distance between the indicator molecules.

[0156] For example, a first fluorescent indicator 320 (e.g., afluorescein derivative) and a second fluorescent indictor 330 (e.g., arhodamine derivative) may be attached to a receptor 300, as depicted inFIG. 8. When no analyte is present short wavelength excitation 310 mayexcite the first fluorescent indicator 320, which fluoresces asindicated by 312. The short wavelength excitation, however, may causelittle or no fluorescence of the second fluorescent indicator 330. Afterbinding of analyte 350 to the receptor, a structural change in thereceptor molecule may bring the first and second fluorescent indicatorscloser to each other. This change in intermolecular distance may allowthe excited first indicator 320 to transfer a portion of its fluorescentenergy 325 to the second fluorescent indicator 330. This transfer inenergy may be measured by either a drop in energy of the fluorescence ofthe first indicator molecule 320, or the detection of increasedfluorescence 314 by the second indicator molecule 330.

[0157] Alternatively, the first and second fluorescent indicators mayinitially be positioned such that short wavelength excitation, may causefluorescence of both the first and second fluorescent indicators, asdescribed above. After binding of analyte 350 to the receptor, astructural change in the receptor molecule may cause the first andsecond fluorescent indicators to move further apart. This change inintermolecular distance may inhibit the transfer of fluorescent energyfrom the first indicator 320 to the second fluorescent indicator 330.This change in the transfer of energy may be measured by either a dropin energy of the fluorescence of the second indicator molecule 330, orthe detection of increased fluorescence by the first indicator molecule320.

[0158] In another embodiment, an indicator ligand may be preloaded ontothe receptor. An analyte may then displace the indicator ligand toproduce a change in the spectroscopic properties of the particles. Inthis case, the initial background absorbance is relatively large anddecreases when the analyte is present. The indicator ligand, in oneembodiment, has a variety of spectroscopic properties which may bemeasured. These spectroscopic properties include, but are not limitedto, ultraviolet absorption, visible absorption, infrared absorption,fluorescence, and magnetic resonance. In one embodiment, the indicatoris a dye having either a strong fluorescence, a strong ultravioletabsorption, a strong visible absorption, or a combination of thesephysical properties. Examples of indicators include, but are not limitedto, carboxyfluorescein, ethidium bromide,7-dimethylamino-4-methylcoumarin, 7-diethylamino-4-methylcoumarin,eosin, erythrosin, fluorescein, Oregon Green 488, pyrene, Rhodamine Red,tetramethylrhodamine, Texas Red, Methyl Violet, Crystal Violet, EthylViolet, Malachite green, Methyl Green, Alizarin Red S, Methyl Red,Neutral Red, o-cresolsulfonephthalein, o-cresolphthalein,phenolphthalein, Acridine Orange, B-naphthol, coumarin, anda-naphthionic acid. When the indicator is mixed with the receptor, thereceptor and indicator interact with each other such that the abovementioned spectroscopic properties of the indicator, as well as otherspectroscopic properties may be altered. The nature of this interactionmay be a binding interaction, wherein the indicator and receptor areattracted to each other with a sufficient force to allow the newlyformed receptor-indicator complex to function as a single unit. Thebinding of the indicator and receptor to each other may take the form ofa covalent bond, an ionic bond, a hydrogen bond, a van der Waalsinteraction, or a combination of these bonds.

[0159] The indicator may be chosen such that the binding strength of theindicator to the receptor is less than the binding strength of theanalyte to the receptor. Thus, in the presence of an analyte, thebinding of the indicator with the receptor may be disrupted, releasingthe indicator from the receptor. When released, the physical propertiesof the indicator may be altered from those it exhibited when bound tothe receptor. The indicator may revert back to its original structure,thus regaining its original physical properties. For example, if afluorescent indicator is attached to a particle that includes areceptor, the fluorescence of the particle may be strong beforetreatment with an analyte containing fluid. When the analyte interactswith the particle, the fluorescent indicator may be released. Release ofthe indicator may cause a decrease in the fluorescence of the particle,since the particle now has less indicator molecules associated with it.

[0160] An example of this type of system is illustrated by the use of aboronic acid substituted resin 505 as a particle. Prior to testing, theboronic acid substituted resin 505 is treated with a sugar 510 which istagged with an indicator (e.g., resorufin) as depicted in FIG. 9. Thesugar 510 binds to the boronic acid receptor 500 imparting a colorchange to the boronic substituted resin 505 (yellow for the resorufintagged sugar). When the boronic acid resin 505 is treated with a fluidsample which includes a sugar 520, the tagged sugar 510 may bedisplaced, causing a decrease in the amount of color produced by theboronic acid substituted resin 505. This decrease may be qualitativelyor, with the use of a detector (e.g., a CCD detector), quantitativelymonitored.

[0161] In another embodiment, a designed synthetic receptor may be used.In one embodiment, a polycarboxylic acid receptor may be attached to apolymeric resin. The polycarboxylic receptors are discussed in U.S.patent application Ser. No. 08/950,712 which is incorporated herein byreference.

[0162] In an embodiment, the analyte molecules in the fluid may bepretreated with an indicator ligand. Pretreatment may involve covalentattachment of an indicator ligand to the analyte molecule. After theindicator has been attached to the analyte, the fluid may be passed overthe sensing particles. Interaction of the receptors on the sensingparticles with the analytes may remove the analytes from the solution.Since the analytes include an indicator, the spectroscopic properties ofthe indicator may be passed onto the particle. By analyzing the physicalproperties of the sensing particles after passage of an analyte stream,the presence and concentration of an analyte may be determined.

[0163] For example, the analytes within a fluid may be derivatized witha fluorescent tag before introducing the stream to the particles. Asanalyte molecules are adsorbed by the particles, the fluorescence of theparticles may increase. The presence of a fluorescent signal may be usedto determine the presence of a specific analyte. Additionally, thestrength of the fluorescence may be used to determine the amount ofanalyte within the stream.

[0164] Receptors

[0165] A variety of natural and synthetic receptors may be used. Thesynthetic receptors may come from a variety of classes including, butnot limited to, polynucleotides (e.g., aptamers), peptides (e.g.,enzymes and antibodies), synthetic receptors, polymeric unnaturalbiopolymers (e.g., polythioureas, polyguanidiniums), and imprintedpolymers., some of which are generally depicted in FIG. 10. Naturalbased synthetic receptors include receptors which are structurallysimilar to naturally occurring molecules. Polynucleotides are relativelysmall fragments of DNA which may be derived by sequentially building theDNA sequence. Peptides may be synthesized from amino acids. Unnaturalbiopolymers are chemical structure which are based on naturalbiopolymers, but which are built from unnatural linking units. Unnaturalbiopolymers such as polythioureas and polyguanidiniums may besynthesized from diamines (i.e., compounds which include at least twoamine functional groups). These molecules are structurally similar tonaturally occurring receptors, (e.g., peptides). Some diamines may, inturn, be synthesized from amino acids. The use of amino acids as thebuilding blocks for these compounds allow a wide variety of molecularrecognition units to be devised. For example, the twenty natural aminoacids have side chains that possess hydrophobic residues, cationic andanionic residues, as well as hydrogen bonding groups. These side chainsmay provide a good chemical match to bind a large number of targets,from small molecules to large oligosaccharides. Amino acid basedpeptides, polythioureas, and polyguanidiniums are depicted in FIG. 10.

[0166] Techniques for the building of DNA fragments and polypeptidefragments on a polymer particle are well known. Techniques for theimmobilization of naturally occurring antibodies and enzymes on apolymeric resin are also well known. The synthesis of polythioureas upona resin particle may be accomplished by the synthetic pathway depictedin FIG. 11. The procedure may begin by deprotection of the terminal tBocprotecting group on an amino acid coupled to a polymeric particle.Removal of the protecting group is followed by coupling of the rigidspacer 410 to the resulting amine 405 using diisopropylcarbodiimide(DIC) and 1-hydroxybenzotriazole hydrate (HOBT). The spacer group mayinhibit formation of a thiazolone by reaction of the first amino acidswith subsequently formed thioureas. After the spacer group is coupled tothe amino acid, another tBoc deprotection is performed to remove thespacer protecting group, giving the amine 415. At this point, monomermay be added incrementally to the growing chain, each time followed by atBoc deprotection. The addition of a derivative of the diamine 420(e.g., an isothiocyanate) to amine 415 gives the mono-thiourea 425. Theaddition of a second thiourea substituent is also depicted. After theaddition of the desired number of monomers, a solution ofbenzylisothiocyanate or acetic anhydride may be added to cap anyremaining amines on the growing oligomers. Between 1 to 20 thioureasgroups may be formed to produce a synthetic polythiourea receptor.

[0167] The synthesis of polyguanidiniums may be accomplished as depictedin FIG. 12. In order to incorporate these guanidinium groups into thereceptor, the coupling of a thiourea with a terminal amine in thepresence of Mukaiyama's reagent may be utilized. The coupling of thefirst thiourea diamine 430 with an amino group of a polymeric particlegives the mono-guanidinium 434. Coupling of the resultingmono-guanidinium with a second thiourea diamine 436 gives adi-guanidinium 438. Further coupling may create a tri-guanidinium 440.Between 1 to 20 guanidinium groups may be formed to produce a syntheticpolyguanidinium receptor.

[0168] The above described methods for making polythioureas andpolyguanidiniums are based on the incorporation of diamines (i.e.,molecules which include at least two amine functional groups) into theoligomeric receptor. The method may be general for any compound havingat least two amino groups. In one embodiment, the diamine may be derivedfrom amino acids. A method for forming diamines from amino acids isshown in FIG. 13. Treatment of a protected amino acid 450 withborane-THF reduces the carboxylic acid portion of the amino acid to theprimary alcohol 452. The primary alcohol is treated with phthalimideunder Mitsunobu conditions (PPh₃/DEAD). The resulting compound 454 istreated with aqueous methylamine to form the desired monoprotecteddiamine 456. The process may be accomplished such that the enantiomericpurity of the starting amino acid is maintained. Any natural orsynthetic amino acid may be used in the above described method.

[0169] The three coupling strategies used to form the respectivefunctional groups may be completely compatible with each other. Thecapability to mix linking groups (amides, thioureas, and guanidiniums)as well as the side chains (hydrophobic, cationic, anionic, and hydrogenbonding) may allow the creation of a diversity in the oligomers that isbeyond the diversity of receptors typically found with naturalbiological receptors. Thus, we may produce ultra-sensitive andultra-selective receptors which exhibit interactions for specifictoxins, bacteria, and environmental chemicals. Additionally, thesesynthetic schemes may be used to build combinatorial libraries ofparticles for use in the sensor array.

[0170] In an embodiment, the indicator ligand may be incorporated intosynthetic receptors during the synthesis of the receptors. The ligandmay be incorporated into a monomeric unit, such as a diamine, that isused during the synthesis of the receptor. In this manner, the indicatormay be covalently attached to the receptor in a controlled position. Byplacing the indicator within the receptor during the synthesis of thereceptor, the positioning of the indicator ligand within the receptormay be controlled. This control may be difficult to achieve aftersynthesis of the receptor is completed.

[0171] In one embodiment, a fluorescent group may be incorporated into adiamine monomer for use in the synthetic sequences. Examples ofmonomeric units which may be used for the synthesis of a receptor aredepicted in FIG. 14. The depicted monomers include fluorescent indicatorgroups. After synthesis, the interaction of the receptor with theanalyte may induce changes in the spectroscopic properties of themolecule. Typically, hydrogen bonding or ionic substituents on thefluorescent monomer involved in analyte binding have the capacity tochange the electron density and/or rigidity of the fluorescent ringsystem, thereby causing observable changes in the spectroscopicproperties of the indicator. For fluorescent indicators such changes maybe exhibited as changes in the fluorescence quantum yield, maximumexcitation wavelength, and/or maximum emission wavelength. This approachdoes not require the dissociation of a preloaded fluorescent ligand,which may be limited in response time by k_((off))). While fluorescentligands are shown here, it is to be understood that a variety of otherligand may be used including calorimetric ligands.

[0172] In another embodiment, two fluorescent monomers for signaling maybe used for the synthesis of the receptor. For example, compound 470 (aderivative of fluorescein) and compound 475 (a derivative of rhodamine),depicted in FIG. 14, may both be incorporated into a synthetic receptor.Compound 470 contains a common colorimetric/fluorescent probe that will,in some embodiments, send out a modulated signal upon analyte binding.The modulation may be due to resonance energy transfer to compound 475.When an analyte binds to the receptor, structural changes in thereceptor may alter the distance between monomeric units 470 and 475. Itis well known that excitation of fluorescein can result in emission fromrhodamine when these molecules are oriented correctly. The efficiency ofresonance energy transfer from monomers 470 to 475 will depend stronglyupon the presence of analyte binding; thus, measurement of rhodaminefluorescence intensity (at a substantially longer wavelength thanfluorescein fluorescence) may serve as an indicator of analyte binding.To greatly improve the likelihood of a modulatory fluorescein-rhodamineinteraction, multiple rhodamine tags may be attached at different sitesalong a receptor molecule without substantially increasing backgroundrhodamine fluorescence (only rhodamine very close to fluorescein willyield appreciable signal). This methodology may be applied to a numberof alternate fluorescent pairs.

[0173] In an embodiment, a large number of chemicalfbiological agents ofinterest to the military and civilian communities may be sensed readilyby the described array sensors including both small and medium sizemolecules. For example, it is known that nerve gases typically producephosphate structures upon hydrolysis in water. The presence of moleculeswhich contain phosphate functional groups may be detected usingpolyguanidiniums. Nerve gases which have contaminated water sources maybe detected by the use of the polyguanidinium receptors described above.

[0174] In order to identify, sense, and quantitate the presence ofvarious bacteria using the proposed micro-machined sensor, twostrategies may be used. First, small molecule recognition and detectionmay be exploited. Since each bacteria possesses a unique and distinctiveconcentration of the various cellular molecules, such as DNA, proteins,metabolites, and sugars, the fingerprint (i.e., the concentration andtypes of DNA, proteins, metabolites, and sugars) of each organism isexpected to be unique. Hence, the analytes obtained from whole bacteriaor broken down bacteria may be used to determine the presence ofspecific bacteria. A series of receptors specific for DNA molecules,proteins, metabolites, and sugars may be incorporated into an array. Asolution containing bacteria, or more preferably broken down bacteria,may be passed over the array of particles. The individual cellularcomponents of the bacteria may interact in a different manner with eachof the particles. This interaction will provide a pattern within thearray which may be unique for the individual bacteria. In this manner,the presence of bacteria within a fluid may be determined.

[0175] In another embodiment, bacteria may be detected as wholeentities, as found in ground water, aerosols, or blood. To detect,sense, and identify intact bacteria, the cell surface of one bacteriamay be differentiated from other bacteria. One method of accomplishingthis differentiation is to target cell surface oligosaccharides (i.e.sugar residues). Each bacterial class (gram negative, gram positive,etc.) displays a different oligosaccharide on their cell surfaces. Theoligosaccharide, which is the code that is read by other cells giving anidentification of the cell, is part of the cell-cell recognition andcommunication process. The use of synthetic receptors which are specificfor oligosaccharides may be used to determine the presence of specificbacteria by analyzing for the cell surface oligosaccharides.

[0176] In another embodiment, the sensor array may be used to optimizewhich receptor molecules should be used for a specific analyte. An arrayof receptors may be placed within the cavities of the supporting memberand a stream containing an analyte may be passed over the array. Thereaction of each portion of the sensing array to the known analyte maybe analyzed and the optimal receptor determined by determining whichparticle, and therefore which receptor, exhibits the strongest reactiontoward the analyte. In this manner, a large number of potentialreceptors may be rapidly scanned. The optimal receptor may then beincorporated into a system used for the detection of the specificanalyte in a mixture of analytes.

[0177] It should be emphasized that although some particles may bepurposefully designed to bind to important species (biological agents,toxins, nerve gasses, etc.), most structures will possess nonspecificreceptor groups. One of the advantages associated with the proposedsensor array is the capacity to standardize each array of particles viaexposure to various analytes, followed by storage of the patterns whicharise from interaction of the analytes with the particles. Therefore,there may not be a need to know the identity of the actual receptor oneach particle. Only the characteristic pattern for each array ofparticles is important. In fact, for many applications it may be lesstime consuming to place the various particles into their respectiveholders without taking precautions to characterize the locationassociated with the specific particles. When used in this manner, eachindividual sensor array may require standardization for the type ofanalyte to be studied.

[0178] On-site calibration for new or unknown toxins may also bepossible with this type of array. Upon complexation of an analyte, thelocal microenvironment of each indicator may change, resulting in amodulation of the light absorption and/or emission properties. The useof standard pattern recognition algorithms completed on a computerplatform may serves as the intelligence factor for the analysis. The“fingerprint” like response evoked from the simultaneous interactionsoccurring at multiple sites within the substrate may be used to identifythe species present in unknown samples.

[0179] The above described sensor array system offers a number ofdistinct advantages over exiting technologies. One advantage is that“real time” detection of analytes may be performed. Another advantage isthat the simultaneous detection of multiple analytes may be realized.Yet another advantage is that the sensor array system allows the use ofsynthetic reagents as well as biologically produced reagents. Syntheticreagents typically have superior sensitivity and specificity towardanalytes when compared to the biological reagents. Yet another advantageis that the sensor array system may be readily modified by simplychanging the particles which are placed within the sensor array. Thisinterchangability may also reduce production costs.

EXAMPLES

[0180] 1. The Determination of pH Using a Chemically Sensitive Particle.

[0181] Shown in FIG. 15 is the magnitude of the optical signaltransmitted through a single polymer particle derivatized witho-cresolphthalein. Here, a filter is used to focus the analysis on thosewavelengths which the dye absorbs most strongly (i.e., about 550 nm).Data is provided for the particle as the pH is cycled between acid andbasic environments. In acidic media (i.e., at times of 100-150 secondsand 180-210 seconds), the particle is clear and the system yields largesignals (up to greater than 300,000 counts) at the optical detector.Between times of 0-100 and 150-180 seconds, the solution was made basic.Upon raising the pH (i.e., making the solution more basic), the particleturns purple in color and the transmitted green light is greatlydiminished. Large signal reductions are recorded under suchcircumstances. The evolution of the signal changes show that theresponse time is quite rapid, on the order of 10 seconds. Furthermore,the behavior is highly reproducible.

[0182] 2. The Simultaneous Detection of Ca⁺², Ce⁺³, and pH by a SensorArray System.

[0183] The synthesis of four different particles was accomplished bycoupling a variety of indictor ligands to a polyethyleneglycol-polystyrene (“PEG-PS”) resin particle. The PEG-PS resin particleswere obtained from Novabiochem Corp., La Jolla, Calif. The particleshave an average diameter of about 130 μm when dry and about 250 μm whenwet. The indicator ligands of fluorescein, o-cresolphthalein complexone,and alizarin complexone were each attached to PEG-PS resin particlesusing a dicyclohexylcarbodiimide (DCC) coupling between a terminal resinbound amine and a carboxylic acid on the indicator ligand.

[0184] These synthetic receptors, localized on the PEG-PS resin tocreate sensing particles, were positioned within micromachined wellsformed in silicon/silicon nitride wafers, thus confining the particlesto individually addressable positions on a multicomponent chip. Thesewells were sized to hold the particles in both swollen and unswollenstates. Rapid introduction of the test fluids can be accomplished usingthese structures while allowing spectrophotometric assays to probe forthe presence of analytes. For the identification and quantification ofanalyte species, changes in the light absorption and light emissionproperties of the immobilized resin particles can be exploited, althoughonly identification based upon absorption properties are discussed here.Upon exposure to analytes, color changes for the particles were found tobe 90% complete within one minute of exposure, although typically onlyseconds were required. To make the analysis of the colorimetric changesefficient, rapid, and sensitive, a charge-coupled-device (CCD) wasdirectly interfaced with the sensor array. Thus, data streams composedof red, green, and blue (RGB) light intensities were acquired andprocessed for each of the individual particle elements. The red, blue,and green responses of the particles to various solutions aregraphically depicted in FIG. 16.

[0185] The true power of the described bead sensor array occurs whensimultaneous evaluation of multiple chemically distinct bead structuresis completed. A demonstration of the capacity of five different beads isprovided in FIG. 16. In this case, blank, alizarin, o-cresol phthalein,fluorescein, and alizarin-Ce3+ complex derivatized beads serve as amatrix for subtle differentiation of chemical environments. The blankbead is simply a polystyrene sphere with no chemical derivatization. Thebead derivatized with o-cresolphthalein responds to Ca+2 at pHs valuesaround 10.0. The binding of calcium is noted from the large green colorattenuation noted for this dye while exposed to the cation. Similarly,the fluorescein derivatized bead acts as a pH sensor. At pHs below 7.4it is light yellow, but at higher pHs it turns dark orange. Interesting,the alizarin complexone plays three distinct roles. First, it acts as aproton sensor yielding a yellow color at pHs below 4.5, orange is notedat pHs between 4.5 and 11.5, and at pHs above 11.5 a blue hue isobserved. Second, it functions as a sensor for lanthanum ions at lowerpHs by turning yellow to orange. Third, the combination of both fluorideand lanthanum ions results in yellow/orange coloration.

[0186] The analysis of solutions containing various amount of Ca⁺² or F⁻at various pH levels was performed using alizarin complexone,o-cresolphthalein complexone, 5-carboxy fluorescein, and alizarin-Ce³⁺complex. A blank particle in which the terminal amines of a PEG-PS resinparticle have been acylated was also used. In this example, the presenceof Ca⁺² (0.1 M Ca(NO₃)₂) was analyzed under conditions of varying pH.The pH was varied to values of 2, 7, and 12, all buffered by a mixtureof 0.04 M phosphate, 0.04 M acetate, and 0.04 M borate. The RGB patternsfor each sensor element in all environments were measured. The beadderivatized with o-cresolphthalein responds to Ca⁺² at pH values around12. Similarly, the 5-carboxy fluorescein derivatized bead acts as a pHsensor. At pHs below 7.4 it is light yellow, but at higher pHs it turnsdark orange. Interesting, the alizarin complexone plays three distinctroles. First, it acts as a proton sensor yielding a yellow color at pHsbelow 4.5, orange is noted at pHs between 4.5 and 11.5, and at pHs above11.5 a blue hue is observed. Second, it functions as a sensor forlanthanum ions at lower pHs by turning yellow to orange. Third, thecombination of both fluoride and lanthanum ions results in yellow/orangecoloration.

[0187] This example demonstrates a number of important factors relatedto the design, testing, and functionality of micromachined array sensorsfor solution analyses. First, derivatization of polymer particles withboth colorimetric and fluorescent dyes was completed. These structureswere shown to respond to pH and Ca²⁺. Second, response times well under1 minute were found. Third, micromachined arrays suitable both forconfinement of particles, as well as optical characterization of theparticles, have been prepared. Fourth, integration of the test bedarrays with commercially available CCD detectors has been accomplished.Finally, simultaneous detection of several analytes in a mixture wasmade possible by analysis of the RGB color patterns created by thesensor array.

[0188] 3. The Detection of Sugar Molecules Using a Boronic Acid BasedReceptor.

[0189] A series of receptors were prepared with functionalities thatassociate strongly with sugar molecules, as depicted in FIG. 9. In thiscase, a boronic acid sugar receptor 500 was utilized to demonstrate thefunctionality of a new type of sensing scheme in which competitivedisplacement of a resorufin derivatized galactose sugar molecule wasused to assess the presence (or lack thereof) of other sugar molecules.The boronic acid receptor 500 was formed via a substitution reaction ofa benzylic bromide. The boronic acid receptor was attached to apolyethylene glycol-polystyrene (“PEG-PS”) resin particle at the “R”position. Initially, the boronic acid derivatized particle was loadedwith resorufin derivatized galactose 510. Upon exposure of the particleto a solution containing glucose 520, the resorufin derivatizedgalactose molecules 510 are displaced from the particle receptor sites.Visual inspection of the optical photographs taken before and afterexposure to the sugar solution show that the boron substituted resin iscapable of sequestering sugar molecules from an aqueous solution.Moreover, the subsequent exposure of the colored particles to a solutionof a non-tagged sugar (e.g., glucose) leads to a displacement of thebound colored sugar reporter molecule. Displacement of this moleculeleads to a change in the color of the particle. The sugar sensor turnsfrom dark orange to yellow in solutions containing glucose. Theparticles were also tested in conditions of varying pH. It was notedthat the color of the particles changes from dark orange to yellow asthe pH is varied from low pH to high pH.

[0190] Further Improvements

[0191] 1. System Improvements

[0192] Shown in FIG. 17 is an embodiment of a system for detectinganalytes in a fluid. In one embodiment, the system includes a lightsource 512, a sensor array 522, a chamber 550 for supporting the sensorarray and a detector 530. The sensor array 522 may include a supportingmember which is configured to hold a variety of particles. In oneembodiment, light originating from the light source 512 passes throughthe sensor array 522 and out through the bottom side of the sensorarray. Light modulated by the particles may be detected by a proximallyspaced detector 530. While depicted as being positioned below the sensorarray, it should be understood that the detector may be positioned abovethe sensor array for reflectance measurements. Evaluation of the opticalchanges may be completed by visual inspection (e.g., by eye, or with theaid of a microscope) or by use of a microprocessor 540 coupled to thedetector.

[0193] In this embodiment, the sensor array 522 is positioned within achamber 550. The chamber 550, may be configured to allow a fluid streamto pass through the chamber such that the fluid stream interacts withthe sensor array 522. The chamber may be constructed of glass (e.g,borosilicate glass or quartz) or a plastic material which is transparentto a portion of the light from the light source. If a plastic materialis used, the plastic material should also be substantially unreactivetoward the fluid. Examples of plastic materials which may be used toform the chamber include, but are not limited to, acrylic resins,polycarbonates, polyester resins, polyethylenes, polyimides, polyvinylpolymers (e.g., polyvinyl chloride, polyvinyl acetate, polyvinyldichloride, polyvinyl fluoride, etc.), polystyrenes, polypropylenes,polytetrafluoroethylenes, and polyurethanes. An example of such achamber is a Sykes-Moore chamber, which is commercially available fromBellco Glass, Inc., in New Jersey. Chamber 550, in one embodiment,includes a fluid inlet port 552 and a fluid outlet port 554. The fluidinlet 552 and outlet 554 ports are configured to allow a fluid stream topass into the interior 556 of the chamber during use. The inlet andoutlet ports may be configured to allow facile placement of a conduitfor transferring the fluid to the chamber. In one embodiment, the portsmay be hollow conduits. The hollow conduits may be configured to have anouter diameter which is substantially equal to the inner diameter of atube for transferring the fluid to or away from the chamber. Forexample, if a plastic or rubber tube is used for the transfer of thefluid, the internal diameter of the plastic tube is substantially equalto the outer diameter of the inlet and outlet ports.

[0194] In another embodiment, the inlet and outlet ports may be Luerlock style connectors. Preferably, the inlet and outlet ports are femaleLuer lock connectors. The use of female Luer lock connectors will allowthe fluid to be introduced via a syringe. Typically, syringes include amale Luer lock connector at the dispensing end of the syringe. For theintroduction of liquid samples, the use of Luer lock connectors mayallow samples to be transferred directly from a syringe to the chamber550. Luer lock connectors may also allow plastic or rubber tubing to beconnected to the chamber using Luer lock tubing connectors.

[0195] The chamber may be configured to allow the passage of a fluidsample to be substantially confined to the interior 556 of the chamber.By confining the fluid to a small interior volume, the amount of fluidrequired for an analysis may be minimized. The interior volume may bespecifically modified for the desired application. For example, for theanalysis of small volumes of fluid samples, the chamber may be designedto have a small interior chamber, thus reducing the amount of fluidneeded to fill the chamber. For larger samples, a larger interiorchamber may be used. Larger chambers may allow a faster throughput ofthe fluid during use.

[0196] In another embodiment, depicted in FIG. 18, a system fordetecting analytes in a fluid includes a light source 512, a sensorarray 522, a chamber 550 for supporting the sensor array and a detector530, all enclosed within a detection system enclosure 560. As describedabove, the sensor array 522 is preferably formed of a supporting memberwhich is configured to hold a variety of particles. Thus, in a singleenclosure, all of the components of an analyte detection system areincluded.

[0197] The formation of an analyte detection system in a singleenclosure may allow the formation of a portable detection system. Forexample, a small controller 570 may be coupled to the analyte detectionsystem. The controller 570 may be configured to interact with thedetector and display the results from the analysis. In one embodiment,the controller includes a display device 572 for displaying informationto a user. The controller may also include input devices 574 (e.g.,buttons) to allow the user to control the operation of the analytedetection system. For example, the controller may control the operationof the light source 512 and the operation of the detector 530.

[0198] The detection system enclosure 560, may be interchangeable withthe controller. Coupling members 576 and 578 may be used to remove thedetection system enclosure 560 from the controller 570. A seconddetection system enclosure may be readily coupled to the controllerusing coupling members 576 and 578. In this manner, a variety ofdifferent types of analytes may be detecting using a variety ofdifferent detection system enclosures. Each of the detection systemenclosures may include different sensor arrays mounted within theirchambers. Instead of having to exchange the sensor array for differenttypes of analysis, the entire detection system enclosure may beexchanged. This may prove advantageous, when a variety of detectionschemes are used. For example a first detection system enclosure may beconfigured for white light applications. The first detection systemenclosure may include a white light source, a sensor that includesparticles that produce a visible light response in the presence of ananalyte, and a detector sensitive to white light. A second detectionsystem enclosure may be configured for fluorescent applications,including a fluorescent light source, a sensor array which includesparticles which produce a fluorescent response on the presence of ananalyte, and a fluorescent detector. The second detection systemenclosure may also include other components necessary for producing aproper detection system. For example, the second detection system mayalso include a filter for preventing short wavelength excitation fromproducing “false” signals in the optical detection system duringfluorescence measurements. A user need only select the proper detectionsystem enclosure for the detection of the desired analyte. Since eachdetection system enclosure includes many of the required components, auser does not have to make light source selections, sensor arrayselections or detector arrangement selections to produce a viabledetection system.

[0199] In another embodiment, the individual components of the systemmay be interchangeable. The system may include coupling members 573 and575 that allow the light source and the detector, respectively, to beremoved from the chamber 550. This may allow a more modular design ofthe system. For example, an analysis may be first performed with a whitelight source to give data corresponding to an absorbance/reflectanceanalysis. After this analysis is performed the light source may bechanged to a ultraviolet light source to allow ultraviolet analysis ofthe particles. Since the particles have already been treated with thefluid, the analysis may be preformed without further treatment of theparticles with a fluid. In this manner a variety of tests may beperformed using a single sensor array.

[0200] In one embodiment, the supporting member is made of any materialcapable of supporting the particles, while allowing the passage of theappropriate wavelength of light. The supporting member may also be madeof a material substantially impervious to the fluid in which the analyteis present. A variety of materials may be used including plastics (e.g.,photoresist materials, acrylic polymers, carbonate polymers, etc.),glass, silicon based materials (e.g., silicon, silicon dioxide, siliconnitride, etc.) and metals. In one embodiment, the supporting memberincludes a plurality of cavities. The cavities are preferably formedsuch that at least one particle is substantially contained within thecavity. Alternatively, a plurality of particles may be contained withina single cavity.

[0201] In some embodiments, it will be necessary to pass liquids overthe sensor array. The dynamic motion of liquids across the sensor arraymay lead to displacement of the particles from the cavities. In anotherembodiment, the particles are preferably held within cavities formed ina supporting member by the use of a transmission electron microscope(“TEM”) grid. As depicted in FIG. 19, a cavity 580 is formed in asupporting member 582. After placement of a particle 584 within thecavity, a TEM grid 586 may be placed atop the supporting member 582 andsecured into position. TEM grids and adhesives for securing TEM grids toa support are commercially available from Ted Pella, Inc., Redding,Calif. The TEM grid 586 may be made from a number of materialsincluding, but not limited to, copper, nickel, gold, silver, aluminum,molybdenum, titanium, nylon, beryllium, carbon, and beryllium-copper.The mesh structure of the TEM grid may allow solution access as well asoptical access to the particles that are placed in the cavities. FIG. 20further depicts a top view of a sensor array with a TEM grid 586 formedupon the upper surface of the supporting member 582. The TEM grid 586may be placed on the upper surface of the supporting member, trappingparticles 584 within the cavities 580. As depicted, the openings 588 inthe TEM grid 586 may be sized to hold the particles 584 within thecavities 580, while allowing fluid and optical access to cavities 580.

[0202] In another embodiment, a sensor array includes a supportingmember configured to support the particles, while allowing the passageof the appropriate wavelength of light to the particle. The supportingmember, in one embodiment, includes a plurality of cavities. Thecavities may be formed such that at least one particle is substantiallycontained within the cavity. The supporting member may be configured tosubstantially inhibit the displacement of the particles from thecavities during use. The supporting member may also be configured toallow the passage of the fluid through cavities, e.g., the fluid mayflow from the top surface of the supporting member, past the particle,and out the bottom surface of the supporting member. This may increasethe contact time between the particle and the fluid.

[0203] FIGS. 21A-G depict a sequence of processing steps for theformation of a silicon based supporting member which includes aremovable top cover and bottom cover. The removable top cover may beconfigured to allow fluids to pass through the top cover and into thecavity. The removable bottom cover may also be configured to allow thefluid to pass through the bottom cover and out of the cavity. Asdepicted in FIG. 21A, a series of layers may be deposited upon bothsides of a silicon substrate 610. First removable layers 612 may bedeposited upon the silicon substrate. The removable layers 612 may besilicon dioxide, silicon nitride, or photoresist material. In oneembodiment, a layer of silicon dioxide 612 is deposited upon bothsurfaces of the silicon substrate 610. Upon these removable layers,covers 614 may be formed. In one embodiment, covers 614 are formed froma material that differs from the material used to form the removablelayers 612 and which is substantially transparent to the light source ofa detection system. For example, if the removable layers 612 are formedfrom silicon dioxide, the cover may be formed from silicon nitride.Second removable layers 616 may be formed upon the covers 614. Secondremovable layers 616 may be formed from a material that differs from thematerial used to form the covers 614. Second removable layers 616 may beformed from a material similar to the material used to form the firstremovable layers 612. In one embodiment, first and second removablelayers 612 and 616 are formed from silicon dioxide and covers 614 areformed from silicon nitride. The layers are patterned and etched usingstandard photolithographic techniques. In one embodiment, the remainingportions of the layers are substantially aligned in the position wherethe cavities are to be formed in the silicon substrate 610.

[0204] After the layers have been etched, spacer structures may beformed on the sidewalls of the first removable layers 612, the covers614, and the second removable layers 616, as depicted in FIG. 21B. Thespacer structures may be formed from the same material used to form thesecond removable layers 616. In one embodiment, depositing a spacerlayer of the appropriate material and subjecting the material to ananisotropic etch may form the spacer structures. An anisotropic etch,such as a plasma etch, employs both physical and chemical removalmechanisms. Ions are typically bombarded at an angle substantiallyperpendicular to the semiconductor substrate upper surface. This causessubstantially horizontal surfaces to be removed faster thansubstantially vertical surfaces. During this etching procedure thespacer layers are preferably removed such that the only regions of thespacer layers that remain may be those regions near substantiallyvertical surfaces, e.g., spacer structures 618.

[0205] After formation of the spacer structures 618, cover supportstructures 620, depicted in FIG. 21C, may be formed. The cover supportstructures may be initially formed by depositing a support structurelayer upon the second removable layer 616 and spacer structures 618. Thesupport structure layer is then patterned and etched, using standardphotolithography, to form the support structures 620. In one embodiment,the support structures are formed from a material that differs from theremovable layers material. In one embodiment, the removable layers maybe formed from silicon dioxide while the support structures and coversmay be formed from silicon nitride.

[0206] Turning to FIG. 21D, the second removable layers 616 and an upperportion of the spacer structures 618 are preferably removed using a wetetch process. Removal of the second removable layers leaves the topsurface of the covers 614 exposed. This allows the covers to bepatterned and etched such that openings 622 are formed extending throughthe covers. These openings 622 may be formed in the covers 614 to allowthe passage of fluid through the cover layers. In one embodiment, theopenings 622 are formed to allow fluid to pass through, while inhibitingdisplacement of the particles from the subsequently formed cavities.

[0207] After the openings 622 have been formed, the remainder of thefirst removable layers 612 and the remainder of the spacer structures618 may be removed using a wet etch. The removal of the removable layersand the spacer structures creates “floating” covers 614, as depicted inFIG. 21E. The covers 614 may be held in proximity to the siliconsubstrate 610 by the support structures 620. The covers 614 may now beremoved by sliding the covers away from the support structures 620. Inthis manner removable covers 614 may be formed.

[0208] After the covers 614 are removed, cavities 640 may be formed inthe silicon substrate 610, as depicted in FIG. 21F. The cavities 640 maybe formed by, initially patterning and etching a photoresist material641 to form a masking layer. After the photoresist material 641 ispatterned, the cavities 640 may be etched into the silicon substrate 610using a hydroxide etch, as described previously.

[0209] After the cavities 640 are formed, the photoresist material maybe removed and particles 642 may be placed within the cavities, asdepicted in FIG. 21G. The particles 642, may be inhibited from beingdisplaced from the cavity 640 by placing covers 614 back onto the upperand lower faces of the silicon substrate 610.

[0210] In another embodiment, a sensor array may be formed using asupporting member, a removable cover, and a secured bottom layer. FIGS.22A-G depict a series of processing steps for the formation of a siliconbased supporting member which includes a removable top cover and asecured bottom layer. The removable top cover is preferably configuredto allow fluids to pass through the top cover and into the cavity. Asdepicted in FIG. 22A, a series of layers may be deposited upon bothsides of a silicon substrate 610. A first removable layer 612 may bedeposited upon the upper face 611 of the silicon substrate 610. Theremovable layer 612 may be silicon dioxide, silicon nitride, orphotoresist material. In one embodiment, a layer of silicon dioxide 612is deposited upon the silicon substrate 610. A cover 614 may be formedupon the removable layer 612 of the silicon substrate 610. In oneembodiment, the cover 614 is formed from a material that differs fromthe material used to form the removable layer 612 and is substantiallytransparent to the light source of a detection system. For example, ifthe removable layer 612 is formed from silicon dioxide, the cover layer614 may be formed from silicon nitride. In one embodiment, a bottomlayer 615 is formed on the bottom surface 613 of the silicon substrate610. In one embodiment, the bottom layer 615 is formed from a materialthat is substantially transparent to the light source of a detectionsystem. A second removable layer 616 may be formed upon the cover 614.Second removable layer 616 may be formed from a material that differsfrom the material used to form the cover layer 614. Second removablelayer 616 may be formed from a material similar to the material used toform the first removable layer 612. In one embodiment, first and secondremovable layers 612 and 616 are formed from silicon dioxide and cover614 is formed from silicon nitride. The layers formed on the uppersurface 611 of the silicon substrate may be patterned and etched usingstandard photolithographic techniques. In one embodiment, the remainingportions of the layers formed on the upper surface are substantiallyaligned in the position where the cavities are to be formed in thesilicon substrate 610.

[0211] After the layers have been etched, spacer structures may beformed on the side walls of the first removable layer 612, the cover614, and the second removable layer 616, as depicted in FIG. 22B. Thespacer structures may be formed from the same material used to form thesecond removable layer 616. In one embodiment, the spacer structures maybe formed by depositing a spacer layer of the appropriate material andsubjecting the spacer layer to an anisotropic etch. During this etchingprocedure the spacer layer is preferably removed such that the onlyregions of the spacer layer which remain may be those regions nearsubstantially vertical surfaces, e.g., spacer structures 618.

[0212] After formation of the spacer structures 618, cover supportstructures 620, depicted in FIG. 22C, may be formed upon the removablelayer 616 and the spacer structures 618. The cover support structures620 may be formed by depositing a support structure layer upon thesecond removable layer 616 and spacer structures 618. The supportstructure layer is then patterned and etched, using standardphotolithography, to form the support structures 620. In one embodiment,the support structures are formed from a material that differs from theremovable layer materials. In one embodiment, the removable layers maybe formed from silicon dioxide while the support structures and covermay be formed from silicon nitride.

[0213] Turning to FIG. 22 D, the second removable layer 616 and an upperportion of the spacer structures 618 may be removed using a wet etchprocess. Removal of the second removable layer leaves the top surface ofthe cover 614 exposed. This allows the cover 614 to be patterned andetched such that openings 622 are formed extending through the cover614. These openings 622 may be formed in the cover 614 to allow thepassage of fluid through the cover. In one embodiment, the openings 622are formed to allow fluid to pass through, while inhibiting displacementof the particle from a cavity. The bottom layer 615 may also besimilarly patterned and etched such that openings 623 may be formedextending thorough the bottom layer 615.

[0214] After the openings 622 and 623 are formed, the first removablelayer 612 and the remainder of the spacer structures 618 may be removedusing a wet etch. The removal of the removable layers and the spacerstructures creates a “floating” cover 614, as depicted in FIG. 22E. Thecover 614 may be held in proximity to the silicon substrate 610 by thesupport structures 620. The cover 614 may now be removed by sliding thecover 614 away from the support structures 620. In this manner aremovable cover 614 may be formed.

[0215] After the cover 614 is removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 22F. The cavities 640 may beformed by, initially patterning and etching a photoresist material 641to form a masking layer. After the photoresist material 614 ispatterned, the cavities 640 may be etched into the silicon substrate 610using a hydroxide etch, as described previously.

[0216] After the cavities 640 are formed, the photoresist material maybe removed and particles 642 may be placed within the cavities, asdepicted in FIG. 22G. The particles 642, may be inhibited from beingdisplaced from the cavity 640 by placing cover 614 back onto the upperface 611 of the silicon substrate 610. The bottom layer 615 may also aidin inhibiting the particle 642 from being displaced from the cavity 640.Openings 622 in cover 614 and openings 623 in bottom layer 615 may allowfluid to pass through the cavity during use.

[0217] In another embodiment, a sensor array may be formed using asupporting member and a removable cover. FIGS. 23A-G depict a series ofprocessing steps for the formation of a silicon based supporting memberwhich includes a removable cover. The removable cover is preferablyconfigured to allow fluids to pass through the cover and into thecavity. As depicted in FIG. 23A, a series of layers may be depositedupon the upper surface 611 of a silicon substrate 610. A first removablelayer 612 may be deposited upon the upper face 611 of the siliconsubstrate 610. The removable layer 612 may be silicon dioxide, siliconnitride, or photoresist material. In one embodiment, a layer of silicondioxide 612 is deposited upon the silicon substrate 610. A cover 614 maybe formed upon the removable layer 612. In one embodiment, the cover isformed from a material which differs from the material used to form theremovable layer 612 and which is substantially transparent to the lightsource of a detection system. For example, if the removable layer 612 isformed from silicon dioxide, the cover 614 may be formed from siliconnitride. A second removable layer 616 may be formed upon the cover 614.Second removable layer 616 may be formed from a material that differsfrom the material used to form the cover 614. Second removable layer 616may be formed from a material similar to the material used to form thefirst removable layer 612. In one embodiment, first and second removablelayers 612 and 616 are formed from silicon dioxide and cover 614 isformed from silicon nitride. The layers formed on the upper surface 611of the silicon substrate may be patterned and etched using standardphotolithographic techniques. In one embodiment, the remaining portionsof the layers formed on the upper surface are substantially aligned inthe position where the cavities are to be formed in the siliconsubstrate 610.

[0218] After the layers have been etched, spacer structures 618 may beformed on the side walls of the first removable layer 612, the coverlayer 614, and the second removable layer 616, as depicted in FIG. 23B.The spacer structures 618 may be formed from the same material used toform the second removable layer 616. In one embodiment, the spacers maybe formed by depositing a spacer layer of the appropriate material uponthe second removable layer and subjecting the material to an anisotropicetch. During this etching procedure the spacer layer is preferablyremoved such that the only regions of the spacer layer which remain maybe those regions near substantially vertical surfaces, e.g., spacerstructures 618.

[0219] After formation of the spacer structures 618, cover supportstructures 620, depicted in FIG. 23C, may be formed upon the removablelayer 616 and the spacer structures 618. The cover support structure maybe formed by initially depositing a support structure layer upon thesecond removable layer 616 and spacer structures 618. The supportstructure layer is then patterned and etched, using standardphotolithography, to form the support structures 620. In one embodiment,the support structures 620 are formed from a material that differs fromthe removable layer materials. In one embodiment, the removable layersmay be formed from silicon dioxide while the support structure and coverlayer may be formed from silicon nitride.

[0220] Turning to FIG. 23D, the second removable layer 616 and an upperportion of the spacer structures 618 may be removed using a wet etchprocess. Removal of the second removable layer leaves the top surface ofthe cover 614 exposed. This allows the cover 614 to be patterned andetched such that openings 622 are formed extending through the cover614. These openings 622 may be formed in the cover 614 to allow thepassage of fluid through the cover 614.

[0221] After the openings 622 are formed, the remainder of the firstremovable layer 612 and the remainder of the spacer structures 618 maybe removed using a wet etch. The removal of the removable layers and thespacer structures creates a “floating” cover 614, as depicted in FIG.23E. The cover 614 is preferably held in proximity to the siliconsubstrate 610 by the support structures 620. The cover 614 may now beremoved by sliding the cover 614 away from the support structures 620.In this manner a removable cover 614 may be formed.

[0222] After the cover 614 is removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 23F. The cavities 640 may beformed by initially depositing and patterning a photoresist material 641upon the silicon support 610. After the photoresist material 614 ispatterned, the cavities 640 may be etched into the silicon substrate 610using a hydroxide etch, as described previously. The etching of thecavities may be accomplished such that a bottom width of the cavity 643is less than a width of a particle 642. In one embodiment, the width ofthe bottom of the cavity may be controlled by varying the etch time.Typically, longer etching times result in a larger opening at the bottomof the cavity. By forming a cavity in this manner, a particle placed inthe cavity may be too large to pass through the bottom of the cavity.Thus, a supporting member that does not include a bottom layer may beformed. An advantage of this process is that the processing steps may bereduced making production simpler.

[0223] After the cavities 640 are formed, the photoresist material maybe removed and particles 642 may be placed within the cavities, asdepicted in FIG. 23G. The particles 642, may be inhibited from beingdisplaced from the cavity 640 by placing cover 614 back onto the upperface 611 of the silicon substrate 610. The narrow bottom portion of thecavity may also aid in inhibiting the particle 642 from being displacedfrom the cavity 640.

[0224] FIGS. 24A-d depict a sequence of processing steps for theformation of a silicon based supporting member which includes a toppartial cover and a bottom partial cover. The top partial cover andbottom partial covers are, in one embodiment, configured to allow fluidsto pass into the cavity and out through the bottom of the cavity. Asdepicted in FIG. 24A, a bottom layer 712 may be deposited onto thebottom surface of a silicon substrate 710. The bottom layer 712 may besilicon dioxide, silicon nitride, or photoresist material. In oneembodiment, a layer of silicon nitride 712 is deposited upon the siliconsubstrate 710. In one embodiment, openings 714 are formed through thebottom layer as depicted in FIG. 24A. Openings 714, in one embodiment,are substantially aligned with the position of the cavities to besubsequently formed. The openings 714 may have a width that issubstantially less than a width of a particle. Thus a particle will beinhibited from passing through the openings 714.

[0225] Cavities 716 may be formed in the silicon substrate 710, asdepicted in FIG. 24B. The cavities 716 may be formed by initiallydepositing and patterning a photoresist layer upon the silicon substrate710. After the photoresist material is patterned, cavities 716 may beetched into the silicon substrate 710 using a number of etchingtechniques, including wet and plasma etches. The width of the cavities716 is preferably greater than the width of a particle, thus allowing aparticle to be placed within each of the cavities. The cavities 716, inone embodiment, are preferably formed such that the cavities aresubstantially aligned over the openings 714 formed in the bottom layer.

[0226] After the cavities have been formed, particles 718 may beinserted into the cavities 716, as depicted in FIG. 24C. The etchedbottom layer 712 may serve as a support for the particles 718. Thus theparticles 718 may be inhibited from being displaced from the cavities bythe bottom layer 712. The openings 714 in the bottom layer 712 may allowfluid to pass through the bottom layer during use.

[0227] After the particles are placed in the cavities, a top layer 720may be placed upon the upper surface 717 of the silicon substrate. Inone embodiment, the top layer 720 is formed from a material issubstantially transparent to the light source of a detection system. Thetop layer may be formed from silicon nitride, silicon dioxide orphotoresist material. In one embodiment, a sheet of photoresist materialis used. After the top layer 620 is formed, openings 719 may be formedin the top layer to allow the passage of the fluid into the cavities. Ifthe top layer 720 is composed of photoresist material, after depositingthe photoresist material across the upper surface of the siliconsubstrate, the openings may be initially formed by exposing thephotoresist material to the appropriate wavelength and pattern of light.If the top layer is compose of silicon dioxide or silicon nitride thetop layer 720 may be developed by forming a photoresist layer upon thetop layer, developing the photoresist, and using the photoresist to etchthe underlying top layer.

[0228] Similar sensor arrays may be produced using materials other thansilicon for the supporting member. For example, as depicted in FIG. 25A-D, the supporting member may be composed of photoresist material. Inone embodiment, sheets of photoresist film may be used to form thesupporting member. Photoresist film sheets are commercially availablefrom E. I. du Pont de Nemours and Company, Wilmington, Del. under thecommercial name RISTON. The sheets come in a variety of sizes, the mostcommon having a thickness ranging from about 1 mil. (25 μm) to about 2mil. (50 μm).

[0229] In an embodiment, a first photoresist layer 722 is developed andetched such that openings 724 are formed. The openings may be formedproximate the location of the subsequently formed cavities. Preferably,the openings have a width that is substantially smaller than a width ofthe particle. The openings may inhibit displacement of the particle froma cavity. After the first photoresist layer 720 is patterned and etched,a main layer 726 is formed upon the bottom layer. The main layer 720 ispreferably formed from a photoresist film that has a thicknesssubstantially greater than a typical width of a particle. Thus, if theparticles have a width of about 30 μm, a main layer may be composed of a50 μm photoresist material. Alternatively, the photoresist layer may becomposed of a multitude of photoresist layers placed upon each otheruntil the desired thickness is achieved, as will be depicted in laterembodiments.

[0230] The main photoresist layer may be patterned and etched to formthe cavities 728, as depicted in FIG. 25B. The cavities, in oneembodiment, are substantially aligned above the previously formedopenings 724. Cavities 728, in one embodiment, have a width which isgreater than a width of a particle.

[0231] For many types of analysis, the photoresist material issubstantially transparent to the light source used. Thus, as opposed toa silicon supporting member, the photoresist material used for the mainsupporting layer may be substantially transparent to the light used bythe light source. In some circumstances, the transparent nature of thesupporting member may allow light from the cavity to migrate, throughthe supporting member, into a second cavity. This leakage of light fromone cavity to the next may lead to detection problems. For example, if afirst particle in a first cavity produces a fluorescent signal inresponse to an analyte, this signal may be transmitted through thesupporting member and detected in a proximate cavity. This may lead toinaccurate readings for the proximately spaced cavities, especially if aparticularly strong signal is produced by the interaction of theparticle with an analyte.

[0232] To reduce the occurrence of this “cross-talk”, a substantiallyreflective layer 730 may be formed along the inner surface of thecavity. In one embodiment, the reflective layer 730 is composed of ametal layer which is formed on the upper surface of the main layer andthe inner surface of the cavity. The metal layer may be deposited usingchemical vapor deposition or other known techniques for depositing thinmetal layers. The presence of a reflective layer may inhibit“cross-talk” between the cavities.

[0233] After the cavities 728 have been formed, particles 718 may beinserted into the cavities 728, as depicted in FIG. 25C. The firstphotoresist layer 722 may serve as a support for the particles 718. Theparticles may be inhibited from being displaced from the cavities by thefirst photoresist layer 722. The openings 724 in the first photoresistlayer 722 may allow fluid to pass through the bottom layer during use.

[0234] After the particles 728 are placed in the cavities 728, a topphotoresist layer 732 may be placed upon the upper surface of thesilicon substrate. After the cover layer is formed, openings 734 may beformed in the cover layer to allow the passage of the fluid into thecavities.

[0235] In another embodiment, the supporting member may be formed from aplastic substrate, as depicted in FIG. 26A-D. In one embodiment, theplastic substrate is composed of a material which is substantiallyresistant to the fluid which includes the analyte. Examples of plasticmaterials which may be used to form the plastic substrate include, butare not limited to, acrylic resins, polycarbonates, polyester resins,polyethylenes, polyimides, polyvinyl polymers (e.g., polyvinyl chloride,polyvinyl acetate, polyvinyl dichloride, polyvinyl fluoride, etc.),polystyrenes, polypropylenes, polytetrafluoroethylenes, andpolyurethanes. The plastic substrate may be substantially transparent orsubstantially opaque to the light produced by the light source. Afterobtaining a suitable plastic material 740, a series of cavities 742 maybe formed in the plastic material. The cavities 740 may be formed bydrilling (either mechanically or with a laser), transfer molding (e.g.,forming the cavities when the plastic material is formed usingappropriately shaped molds), or using a punching apparatus to punchcavities into the plastic material. In one embodiment, the cavities 740are formed such that a lower portion 743 of the cavities issubstantially narrower than an upper portion 744 of the cavities. Thelower portion 743 of the cavities may have a width substantially lessthan a width of a particle. The lower portion 743 of the cavities 740may inhibit the displacement of a particle from the cavity 740. Whiledepicted as rectangular, with a narrower rectangular opening at thebottom, it should be understood that the cavity may be formed in anumber of shapes including but not limited to pyramidal, triangular,trapezoidal, and oval shapes. An example of a pyramidal cavity which istapered such that the particle is inhibited from being displaced fromthe cavity is depicted in FIG. 25D.

[0236] After the cavities 742 are formed, particles 718 may be insertedinto the cavities 742, as depicted in FIG. 26B. The lower portion 743 ofthe cavities may serve as a support for the particles 718. The particles718 may be inhibited from being displaced from the cavities 742 by thelower portion 743 of the cavity. After the particles are placed in thecavities 740, a cover 744 may be placed upon the upper surface 745 ofthe plastic substrate 740, as depicted in FIG. 26C. In one embodiment,the cover is formed from a film of photoresist material. After the cover744 is placed on the plastic substrate 740, openings 739 may be formedin the cover layer to allow the passage of the fluid into the cavities.

[0237] In some circumstances a substantially transparent plasticmaterial may be used. As described above, the use of a transparentsupporting member may lead to “cross-talk” between the cavities. Toreduce the occurrence of this “cross-talk”, a substantially reflectivelayer 748 may be formed on the inner surface 746 of the cavity, asdepicted in FIG. 26E. In one embodiment, the reflective layer 748 iscomposed of a metal layer which is formed on the inner surface of thecavities 742. The metal layer may be deposited using chemical vapordeposition or other techniques for depositing thin metal layers. Thepresence of a reflective layer may inhibit cross-talk between thecavities.

[0238] In another embodiment, a silicon based supporting member for asensing particle may be formed without a bottom layer. In thisembodiment, the cavity may be tapered to inhibit the passage of theparticle from the cavity, through the bottom of the supporting member.FIG. 27A-D, depicts the formation of a supporting member from a siliconsubstrate. In this embodiment, a photoresist layer 750 is formed upon anupper surface of a silicon substrate 752, as depicted in FIG. 27A. Thephotoresist layer 750 may be patterned and developed such that theregions of the silicon substrate in which the cavities will be formedare exposed.

[0239] Cavities 754 may now be formed, as depicted in FIG. 27B, bysubjecting the silicon substrate to an anisotropic etch. In oneembodiment, a potassium hydroxide etch is used to produced taperedcavities. The etching may be controlled such that the width of thebottom of the cavities 750 is less than a width of the particle. Afterthe cavities have been etched, a particle 756 may be inserted into thecavities 754 as depicted in FIG. 27C. The particle 756 may be inhibitedfrom passing out of the cavities 754 by the narrower bottom portion ofthe cavities. After the particle is positioned within the cavities 754,a cover 758 may be formed upon the silicon substrate 752, as depicted inFIG. 27D. The cover may be formed of any material substantiallytransparent to the light produced by the light source used for analysis.Openings 759 may be formed in the cover 758 to allow the fluid to passinto the cavity from the top face of the supporting member 752. Theopenings 759 in the cover and the opening at the bottom of the cavities754 together may allow fluid to pass through the cavity during use.

[0240] In another embodiment, a supporting member for a sensing particlemay be formed from a plurality of layers of a photoresist material. Inthis embodiment, the cavity may be tapered to inhibit the passage of theparticle from the cavity, through the bottom of the supporting member.FIGS. 28A-E depict the formation of a supporting member from a pluralityof photoresist layers. In an embodiment, a first photoresist layer 760is developed and etched to form a series of openings 762 which arepositioned at the bottom of subsequently formed cavities, as depicted inFIG. 28A. As depicted in FIG. 28B, a second layer of photoresistmaterial 764 may be formed upon the first photoresist layer 760. Thesecond photoresist layer may be developed and etched to form openingssubstantially aligned with the openings of the first photoresist layer760. The openings formed in the second photoresist layer 764, in oneembodiment, are substantially larger than the layers formed in the firstphotoresist layer 760. In this manner, a tapered cavity may be formedwhile using multiple photoresist layers.

[0241] As depicted in FIG. 28C, additional layers of photoresistmaterial 766 and 768 may be formed upon the second photoresist layer764. The openings of the additional photoresist layers 766 and 768 maybe progressively larger as each layer is added to the stack. In thismanner, a tapered cavity may be formed. Additional layers of photoresistmaterial may be added until the desired thickness of the supportingmember is obtained. The thickness of the supporting member, in oneembodiment, is greater than a width of a particle. For example, if alayer of photoresist material has a thickness of about 25 μm and aparticle has a width of about 100 μm, a supporting member may be formedfrom four or more layers of photoresist material. While depicted aspyramidal, the cavity may be formed in a number of different shapes,including but not limited to, rectangular, circular, oval, triangular,and trapezoidal. Any of these shapes may be obtained by appropriatepatterning and etching of the photoresist layers as they are formed.

[0242] In some instances, the photoresist material may be substantiallytransparent to the light produced by the light source. As describedabove, the use of a transparent supporting member may lead to“cross-talk” between the cavities. To reduce the occurrence of this“cross-talk”, a substantially reflective layer 770 may be formed alongthe inner surface of the cavities 762, as depicted in FIG. 28D. In oneembodiment, the reflective layer is composed of a metal layer which isformed on the inner surface of the cavities 762. The metal layer may bedeposited using chemical vapor deposition or other techniques fordepositing thin metal layers. The presence of a reflective layer mayinhibit “cross-talk” between the cavities.

[0243] After the cavities 762 are formed, particles 772 may be insertedinto the cavities 762, as depicted in FIG. 28D. The narrow portions ofthe cavities 762 may serve as a support for the particles 772. Theparticles 772 may be inhibited from being displaced from the cavities762 by the lower portion of the cavities. After the particles 772 areplaced in the cavities 762, a cover 774 may be placed upon the uppersurface of the top layer 776 of the supporting member, as depicted inFIG. 28E. In one embodiment, the cover 774 is also formed from a film ofphotoresist material. After the cover layer is formed, openings 778 maybe formed in the cover 774 to allow the passage of the fluid into thecavities.

[0244] In another embodiment, a supporting member for a sensing particlemay be formed from photoresist material which includes a particlesupport layer. FIGS. 29A-E depict the formation of a supporting memberfrom a series of photoresist layers. In an embodiment, a firstphotoresist layer 780 is developed and etched to form a series ofopenings 782 which may become part of subsequently formed cavities. Inanother embodiment, a cavity having the appropriate depth may be formedby forming multiple layers of a photoresist material, as describedpreviously. As depicted in FIG. 29B, a second photoresist layer 784 maybe formed upon the first photoresist layer 780. The second photoresistlayer 784 may be patterned to form openings substantially aligned withthe openings of the first photoresist layer 782. The openings formed inthe second photoresist layer 784 may be substantially equal in size tothe previously formed openings. Alternatively, the openings may bevariable in size to form different shaped cavities.

[0245] For reasons described above, a substantially reflective layer 786may be formed along the inner surface of the cavities 782 and the uppersurface of the second photoresist layer 784, as depicted in FIG. 29C. Inone embodiment, the reflective layer is composed of a metal layer. Themetal layer may be deposited using chemical vapor deposition or othertechniques for depositing thin metal layers. The presence of areflective layer may inhibit “cross-talk” between the cavities.

[0246] After the metal layer is deposited, a particle support layer 788may be formed on the bottom surface of the first photoresist layer 780,as depicted in FIG. 29D. The particle support layer 788 may be formedfrom photoresist material, silicon dioxide, silicon nitride, glass or asubstantially transparent plastic material. The particle support layer788 may serve as a support for the particles placed in the cavities 782.The particle support layer, in one embodiment, is formed from a materialthat is substantially transparent to the light produced by the lightsource.

[0247] After the particle supporting layer 788 is formed, particles 785may be inserted into the cavities 782, as depicted in FIG. 29E. Theparticle support layer 788 may serve as a support for the particles.Thus the particles 785 may be inhibited from being displaced from thecavities by the particle support layer 788. After the particles 785 areplaced in the cavities 782, a cover 787 may be placed upon the uppersurface of the second photoresist layer 784, as depicted in FIG. 29E. Inone embodiment, the cover is also formed from a film of photoresistmaterial. After the cover is formed, openings 789 may be formed in thecover 787 to allow the passage of the fluid into the cavities. In thisembodiment, the fluid is inhibited from flowing through the supportingmember. Instead, the fluid may flow into and out of the cavities via theopenings 789 formed in the cover 787.

[0248] A similar supporting member may be formed from a plasticmaterial, as depicted in FIGS. 30A-D. The plastic material may besubstantially resistant to the fluid which includes the analyte. Theplastic material may be substantially transparent or substantiallyopaque to the light produced by the light source. After obtaining asuitable plastic substrate 790, a series of cavities 792 may be formedin the plastic substrate 790. The cavities may be formed by drilling(either mechanically or with a laser), transfer molding (e.g., formingthe cavities when the plastic substrate is formed using appropriatelyshaped molds), or using a punching machine to form the cavities. In oneembodiment, the cavities extend through a portion of the plasticsubstrate, terminating proximate the bottom of the plastic substrate,without passing through the plastic substrate. After the cavities 792are formed, particles 795 may be inserted into the cavities 792, asdepicted in FIG. 30B. The bottom of the cavity may serve as a supportfor the particles 795. After the particles are placed in the cavities, acover 794 may be placed upon the upper surface of the plastic substrate790, as depicted in FIG. 30C. In one embodiment, the cover may be formedfrom a film of photoresist material. After the cover 794 is formed,openings 796 may be formed in the cover to allow the passage of thefluid into the cavities. While depicted as rectangular, is should beunderstood that the cavities may be formed in a variety of differentshapes, including triangular, pyramidal, pentagonal, polygonal, oval, orcircular. It should also be understood that cavities having a variety ofdifferent shapes may be formed into the same plastic substrate, asdepicted in FIG. 30D.

[0249] In one embodiment, a series of channels may be formed in thesupporting member interconnecting some of the cavities, as depicted inFIG. 3. Pumps and valves may also be incorporated into the supportingmember to aid passage of the fluid through the cavities. A schematicfigure of a diaphragm pump 800 is depicted in FIG. 31. Diaphragm pumps,in general, include a cavity 810, a flexible diaphragm 812, an inletvalve 814, and an outlet valve 816. The flexible diaphragm 812, duringuse, is deflected as shown by arrows 818 to create a pumping force. Asthe diaphragm is deflected toward the cavity 810 it may cause the inletvalve 814 to close, the outlet valve 816 to open and any liquid which isin the cavity 810 will be forced toward the outlet 816. As the diaphragmmoves away from the cavity 810, the outlet valve 816 may be pulled to aclosed position, and the inlet valve 814 may be opened, allowingadditional fluid to enter the cavity 810. In this manner a pump may beused to pump fluid through the cavities. It should be understood thatthe pump depicted in FIG. 31 is a generalized version of a diaphragmbased pump. Actual diaphragm pumps may have different shapes or may haveinlet and outlet valves which are separate from the pumping device.

[0250] In one embodiment, the diaphragm 810 may be made from apiezoelectric material. This material will contract or expand when anappropriate voltage is applied to the diaphragm. Pumps using apiezoelectric diaphragms are described in U.S. Pat. Nos. 4,344,743,4,938,742, 5,611,676, 5,705,018, and 5,759,015, all of which areincorporated herein by reference. In other embodiments, the diaphragmmay be activated using a pneumatic system. In these systems, an airsystem may be coupled to the diaphragm such that changes in air densityabout the diaphragm, induced by the pneumatic system, may cause thediaphragm to move toward and away from the cavity. A pneumaticallycontrolled pump is described in U.S. Pat. No. 5,499,909 which isincorporated herein by reference. The diaphragm may also be controlledusing a heat activated material. The diaphragm may be formed from atemperature sensitive material. In one embodiment, the diaphragm may beformed from a material which is configured to expand and contract inresponse to temperature changes. A pump system which relies ontemperature activated diaphragm is described in U.S. Pat. No. 5,288,214which is incorporated herein by reference.

[0251] In another embodiment, an electrode pump system may be used. FIG.32 depicts a typical electrode based system. A series of electrodes 820may be arranged along a channel 822 which may lead to a cavity 824 whichincludes a particle 826. By varying the voltage in the electrodes 820 acurrent flow may be induced in the fluid within the channel 822.Examples of electrode based systems include, but are not limited to,electroosmosis systems, electrohydrodynamic systems, and combinations ofelectroosmosis and electrohydrodynamic systems.

[0252] Electrohydrodynamic pumping of fluids is known and may be appliedto small capillary channels. In an electrohydrodynamic system electrodesare typically placed in contact with the fluid when a voltage isapplied. The applied voltage may cause a transfer in charge either bytransfer or removal of an electron to or from the fluid. This electrontransfer typically induces liquid flow in the direction from thecharging electrode to the oppositely charged electrode.Electrohydrodynamic pumps may be used for pumping fluids such as organicsolvents.

[0253] Electroosmosis, is a process which involves applying a voltage toa fluid in a small space, such as a capillary channel, to cause thefluid to flow. The surfaces of many solids, including quartz, glass andthe like, become variously charged, negatively or positively, in thepresence of ionic materials, such as for example salts, acids or bases.The charged surfaces will attract oppositely charged (positive ornegative) counterions in aqueous solutions. The application of a voltageto such a solution results in a migration of the counterions to theoppositely charged electrode, and moves the bulk of the fluid as well.The volume flow rate is proportional to the current, and the volume flowgenerated in the fluid is also proportional to the applied voltage. Anelectroosmosis pump system is described in U.S. Pat. No. 4,908,112 whichis incorporated herein by reference.

[0254] In another embodiment, a combination of electroosmosis pumps andelectrohydrodynamic pumps may be used. Wire electrodes may be insertedinto the walls of a channel at preselected intervals to form alternatingelectroosmosis and electrohydrodynamic devices. Because electroosmosisand electrohydrodynamic pumps are both present, a plurality of differentsolutions, both polar and non-polar, may be pump along a single channel.Alternatively, a plurality of different solutions may be passed along aplurality of different channels connected to a cavity. A system whichincludes a combination of electroosmosis pumps and electrohydrodynamicpumps is described in U.S. Pat. No. 5,632,876 which is incorporatedherein by reference.

[0255] In an embodiment, a pump may be incorporated into a sensor arraysystem, as depicted in FIG. 32. A sensor array 830 includes at least onecavity 832 in which a particle 834 may be placed. The cavity 832 may beconfigured to allow fluid to pass through the cavity during use. A pump836 may be incorporated onto a portion of the supporting member 838. Achannel 831 may be formed in the supporting member 838 coupling the pump836 to the cavity 832. The channel 831 may be configured to allow thefluid to pass from the pump 836 to the cavity 832. The pump 836 may bepositioned away from the cavity 832 to allow light to be directedthrough the cavity during use. The supporting member 838 and the pump836 may be formed from a silicon substrate, a plastic material, orphotoresist material. The pump 836 may be configured to pump fluid tothe cavity via the channel, as depicted by the arrows in FIG. 32. Whenthe fluid reaches the cavity 832, the fluid may flow past the particle834 and out through the bottom of the cavity. An advantage of usingpumps is that better flow through the channels may be achieved.Typically, the channels and cavities may have a small volume. The smallvolume of the cavity and channel tends to inhibit flow of the fluidthrough the cavity. By incorporating a pump, the flow of fluid to thecavity and through the cavity may be increased, allowing more rapidtesting of the fluid sample. While a diaphragm based pump system isdepicted in FIG. 33, it should be understood that electrode basedpumping systems may also be incorporated into the sensor array toproduce fluid flows.

[0256] In another embodiment, a pump may be coupled to a supportingmember for analyzing analytes in a fluid stream, as depicted in FIG. 34.A channel 842 may couple a pump 846 to multiple cavities 844 formed in asupporting member 840. The cavities 842 may include sensing particles848. The pump may be configured to create a flow of the fluid throughthe channel 842 to the cavities 848. In one embodiment, the cavities mayinhibit the flow of the fluid through the cavities 844. The fluid mayflow into the cavities 844 and past the particle 848 to create a flow offluid through the sensor array system. In this manner a single pump maybe used to pass the fluid to multiple cavities. While a diaphragm pumpsystem is depicted in FIG. 33, it should be understood that electrodepumping systems may also be incorporated into the supporting member tocreate similar fluid flows.

[0257] In another embodiment, multiple pumps may be coupled to asupporting member of a sensor array system. In one embodiment, the pumpsmay be coupled in series with each other to pump fluid to each of thecavities. As depicted in FIG. 35, a first pump 852 and a second pump 854may be coupled to a supporting member 850. The first pump 852 may becoupled to a first cavity 856. The first pump may be configured totransfer fluid to the first cavity 856 during use. The cavity 856 may beconfigured to allow the fluid to pass through the cavity to a firstcavity outlet channel 858. A second pump 854 may also be coupled to thesupporting member 850. The second pump 854 may be coupled to a secondcavity 860 and the first cavity outlet channel 858. The second pump 854may be configured to transfer fluid from the first cavity outlet channel858 to the second cavity 860. The pumps may be synchronized such that asteady flow of fluid through the cavities is obtained. Additional pumpsmay be coupled to the second cavity outlet channel 862 such that thefluid may be pumped to additional cavities. In one embodiment, each ofthe cavities in the supporting member is coupled to a pump configured topump the fluid stream to the cavity.

[0258] In another embodiment, multiple electrode based pumps may beincorporated herein into the sensor array system. The pumps may beformed along the channels which couple the cavities. As depicted in FIG.36, a plurality of cavities 870 may be formed in a supporting member 872of a sensor array. Channels 874 may also be formed in the supportingmember 872 interconnecting the cavities 870 with each other. An inletchannel 876 and an outlet channel 877, which allow the fluid to passinto and out of the sensor array, respectively, may also be formed. Aseries of electrodes 878 may be positioned over the channels 874, 876,and 877. The electrodes may be used to form an electroosmosis pumpingsystem or an electrohydrodynamic pumping system. The electrodes may becoupled to a controller 880 which may apply the appropriate voltage tothe appropriate electrodes to produce a flow of the fluid through thechannels. The pumps may be synchronized such that a steady flow of fluidthrough the cavities is obtained. The electrodes may be positionedbetween the cavities such that the electrodes do not significantlyinterfere with the application of light to the cavities.

[0259] In some instances it may be necessary to add a reagent to aparticle before, during or after an analysis process. Reagents mayinclude receptor molecules or indicator molecules. Typically, suchreagents may be added by passing a fluid stream which includes thereagent over the sensor array. In an embodiment, the reagent may beincorporated herein into the sensor array system which includes twoparticles. In this embodiment, a sensor array system 900 may include twoparticles 910 and 920 for each sensing position of the sensor array, asdepicted in FIG. 37. The first particle 910 may be positioned in a firstcavity 912. The second particle 920 may be positioned in a second cavity922. In one embodiment, the second cavity is coupled to the first cavityvia a channel 930. The second particle includes a reagent which is atleast partially removable from the second particle 920. The reagent mayalso be configured to modify the first particle 910, when the reagent iscontacted with the first particle, such that the first particle willproduce a signal when the first particle interacts with an analyteduring use. The reagent may be added to the first cavity before, duringor after a fluid analysis. The reagent is preferably coupled to thesecond particle 920. The a portion of the reagent coupled to the secondparticle may be decoupled from the particle by passing a decouplingsolution past the second particle. The decoupling solution may include adecoupling agent which will cause at least a portion of the reagent tobe at released by the particle. A reservoir 940 may be formed on thesensor array to hold the decoupling solution.

[0260] A first pump 950 and a second pump 960 may also be coupled to thesupporting member 915. The first pump 950 may be configured to pumpfluid from a fluid inlet 952 to the first cavity 912 via channel 930.The fluid inlet 952 is the location where the fluid, which includes theanalyte, is introduced into the sensor array system. A second pump 950may be coupled to the reservoir 940 and the second cavity 922. Thesecond pump 960 may be used to transfer the decoupling solution from thereservoir to the second cavity 922. The decoupling solution may passthrough the second cavity 922 and into first cavity 912. Thus, as thereagent is removed the second particle it may be transferred to thefirst cavity912, where the reagent may interact with the first particle910. The reservoir may be refilled by removing the reservoir outlet 942,and adding additional fluid to the reservoir 940. While diaphragm basedpump systems are depicted in FIG. 37, it should be understood thatelectrode based pumping systems may also be incorporated herein into thesensor array to produce fluid flows.

[0261] The use of such a system is described by way of example. In someinstances it may be desirable to add a reagent to the first particleprior to passing the fluid which includes the analyte to the firstparticle. The reagent may be coupled to the second particle and placedin the sensor array prior to use, typically during construction of thearray. A decoupling solution may be added to the reservoir before use. Acontroller 970 may also be coupled to the system to allow automaticoperation of the pumps. The controller 970 may be configured to initiatethe analysis sequence by activating the second pump 960, causing thedecoupling solution to flow from the reservoir 940 to the second cavity922. As the fluid passes through the second cavity 922, the decouplingsolution may cause at least some of the reagent molecules to be releasedfrom the second particle 920. The decoupling solution may be passed outof the second cavity 922 and into the first cavity 912. As the solutionpasses through the first cavity, some of the reagent molecules may becaptured by the first particle 910. After a sufficient number ofmolecules have been captured by the first particle 910, flow of fluidthorough the second cavity 922 may be stopped. During thisinitialization of the system, the flow of fluid through the first pumpmay be inhibited.

[0262] After the system is initialized, the second pump may be stoppedand the fluid may be introduced to the first cavity. The first pump maybe used to transfer the fluid to the first cavity. The second pump mayremain off, thus inhibiting flow of fluid from the reservoir to thefirst cavity. It should be understood that the reagent solution may beadded to the first cavity while the fluid is added to the first cavity.In this embodiment, both the first and second pumps may be operatedsubstantially simultaneously.

[0263] Alternatively, the reagent may be added after an analysis. Insome instances, a particle may interact with an analyte such that achange in the receptors attached to the first particle occurs. Thischange may not, however produce a detectable signal. The reagentattached to the second bead may be used to produce a detectable signalwhen it interacts with the first particle, if a specific analyte ispresent. In this embodiment, the fluid is introduced into the cavityfirst. After the analyte has been given time to react with the particle,the reagent may be added to the first cavity. The interaction of thereagent with the particle may produce a detectable signal. For example,an indicator reagent may react with a particle which has been exposed toan analyte to produce a color change on the particle. Particle whichhave not been exposed to the analyte may remain unchanged or show adifferent color change.

[0264] As shown in FIG. 1, a system for detecting analytes in a fluidmay include a light source 110, a sensor array 120 and a detector 130.The sensor array 120 is preferably formed of a supporting member whichis configured to hold a variety of particles 124 in an ordered array. Ahigh sensitivity CCD array may be used to measure changes in opticalcharacteristics which occur upon binding of the biological/chemicalagents. Data acquisition and handling is preferably performed withexisting CCD technology. As described above, colorimetric analysis maybe performed using a white light source and a color CCD detector.However, color CCD detectors are typically more expensive than grayscale CCD detectors.

[0265] In one embodiment, a gray scale CCD detector may be used todetect colorimetric changes. In one embodiment, a gray scale detectormay be disposed below a sensor array to measure the intensity of lightbeing transmitted through the sensor array. A series of lights (e.g.,light emitting diodes) may be arranged above the sensor array. In oneembodiment, groups of three LED lights may be arranged above each of thecavities of the array. Each of these groups of LED lights may include ared, blue and a green light. Each of the lights may be operatedindividually such that one of the lights may be on while the other twolights are off. In order to provide color information while using a grayscale detector, each of the lights is sequentially turned on and thegray scale detector is used to measure the intensity of the lightpassing through the sensor array. After information from each of thelights is collected, the information may be processed to derive theabsorption changes of the particle.

[0266] In one embodiment, the data collected by the gray scale detectormay be recorded using 8 bits of data. Thus, the data will appear as avalue between 0 and 255. The color of each chemical sensitive elementmay be represented as a red, blue and green value. For example, a blankparticle (i.e., a particle which does not include a receptor) willtypically appear white. When each of the LED lights (red, blue andgreen) are operated the CCD detector will record a value correspondingto the amount of light transmitted through the cavity. The intensity ofthe light may be compared to a blank particle, to determine theabsorbance of a particle with respect to the LED light which is used.Thus, the red, green and blue components may be recorded individuallywithout the use of a color CCD detector. In one embodiment, it is foundthat a blank particle exhibits an absorbance of about 253 whenilluminated with a red LED, a value of about 250 when illuminated by agreen LED, and a value of about 222 when illuminated with a blue LED.This signifies that a blank particle does not significantly absorb red,green or blue light. When a particle with a receptor is scanned, theparticle may exhibit a color change, due to absorbance by the receptor.For example, it was found that when a particle which includes a5-carboxyfluorescein receptor is subjected to white light, the particleshows a strong absorbance of blue light. When a red LED is used toilluminate the particle, the gray scale CCD detector may detect a valueof about 254. When the green LED is used, the gray scale detector maydetect a value of about 218. When a blue LED light is used, a gray scaledetector may detect a value of about 57. The decrease in transmittanceof blue light is believed to be due to the absorbance of blue light bythe 5-carboxyfluorescein. In this manner the color changes of a particlemay be quantitatively characterized using a gray scale detector.

[0267] As described above, after the cavities are formed in thesupporting member, a particle may be positioned at the bottom of acavity using a micromanipulator. This allows the location of aparticular particle to be precisely controlled during the production ofthe array. The use of a micromanipulator may, however, be impracticalfor production of sensor array systems. An alternate method of placingthe particles into the cavities may involve the use of a silk screenlike process. A series of masking materials may be placed on the uppersurface of the sensor array prior to filling the cavities. The maskingmaterials may be composed of glass, metal or plastic materials. Acollection of particles may be placed upon the upper surface of themasking materials and the particles may be moved across the surface.When a cavity is encountered, a particle may drop into the cavity if thecavity is unmasked. Thus particles of known composition are placed inonly the unmasked regions. After the unmasked cavities are filled, themasking pattern may be altered and a second type of particles may bespread across the surface. Preferably, the masking material will maskthe cavities that have already been filled with particle. The maskingmaterial may also mask other non-filled cavities. This technique may berepeated until all of the cavities are filled. After filling thecavities, a cover may be placed on the support member, as describedabove, to inhibit the displacement and mixing of the particles. Anadvantage of such a process is that it may be more amenable toindustrial production of supporting members.

[0268] 2. Further System Improvements

[0269] One challenge in a chemical sensor system is keeping dead volumeto a minimum. This is especially problematic when an interface to theoutside world is required (e.g., a tubing connection). In many cases the“dead volume” associated with the delivery of the sample to the reactionsite in a “lab-on-a-chip” may far exceed the actual amount of reagentrequired for the reaction. Filtration is also frequently necessary toprevent small flow channels in the sensor arrays from plugging. Here thefilter can be made an integral part of the sensor package.

[0270] In an embodiment, a system for detecting an analyte in a fluidincludes a conduit coupled to a sensor array and a vacuum chambercoupled to the conduit. FIG. 38 depicts a system in which a fluid stream(E) passes through a conduit (D), onto a sensor array (G), and into avacuum apparatus (F). The vacuum apparatus (F) may be coupled to theconduit (D) downstream from the sensor array (G). A vacuum apparatus isherein defined to be any system capable of creating or maintaining avolume at a pressure below atmospheric. Examples of vacuum apparatusinclude vacuum chambers. Vacuum chamber, in one embodiment, may besealed tubes from which a portion of the air has been evacuated,creating a vacuum within the tube. A commonly used example of such asealed tube is a “vacutainer” system commercially available from BectonDickinson. Alternatively, a vacuum chamber which is sealed by a movablepiston may also be used to generate a vacuum. For example, a syringe maybe coupled to the conduit. Movement of the piston (i.e., the plunger)away from the chamber will create a partial vacuum within the chamber.Alternatively, the vacuum apparatus may be a vacuum pump or vacuum line.Vacuum pumps may include direct drive pumps, oil pumps, aspirator pumpsor micropumps. Micropumps that may be incorporated into a sensor arraysystem have been previously described.

[0271] As opposed to previously described methods, in which a pump asused to force a fluid stream through a sensor array, the use of a vacuumapparatus allows the fluid to be pulled through the sensor array.Referring to FIG. 39, the vacuum apparatus (F) is coupled to downstreamfrom a sensor array. When coupled to the conduit (D), the vacuumapparatus may exert a suction force on the fluid stream, forcing aportion of the stream to pass over, and in some instances, through thesensor array. In some embodiments, the fluid may continue to passthrough the conduit, after passing the sensor array, and into the vacuumapparatus. In an embodiment where the vacuum apparatus is apre-evacuated tube, the fluid flow will continue until the air withinthe tube is at a pressure substantially equivalent to the atmosphericpressure. The vacuum apparatus may include a penetrable wall (H). Thepenetrable wall forms a seal inhibiting air from entering the vacuumapparatus. When the wall is broken or punctured, air from outside of thesystem will begin to enter the vacuum apparatus. In one embodiment, theconduit includes a penetrating member, (e.g., a syringe needle), whichallows the penetrable wall to be pierced. Piercing the penetrable wallcauses air and fluid inside the conduit to be pulled through the conduitinto the vacuum apparatus until the pressure between the vacuumapparatus and the conduit is equalized.

[0272] The sensor array system may also include a filter (B) coupled tothe conduit (D) as depicted in FIG. 39. The filter (B) may be positionedalong the conduit, upstream from the sensor array. Filter (B) may be aporous filter which includes a membrane for removing components from thefluid stream. In one embodiment, the filter may include a membrane forremoval of particulates above a minimum size. The size of theparticulates removed will depend on the porosity of the membrane as isknown in the art. Alternatively, the filter may be configured to removeunwanted components of a fluid stream. For example, if the fluid streamis a blood sample, the filter may be configured to remove red and whiteblood cells from the stream, while leaving in the blood stream bloodplasma and other components therein.

[0273] The sensor array may also include a reagent delivery reservoir(C). The reagent delivery system is preferably coupled to the conduitupstream from the sensor array. The reagent delivery reservoir may beformed from a porous material which includes a reagent of interest. Asthe fluid passes through this reservoir, a portion of the reagent withinthe regent delivery reservoir passes into the fluid stream. The fluidreservoir may include a porous polymer or filter paper on which thereagent is stored. Examples of reagents which may be stored within thereagent delivery reservoir include, but are not limited to,visualization agents (e.g., dye or fluorophores), co-factors, buffers,acids, bases, oxidants, and reductants.

[0274] The sensor array may also include a fluid sampling device (A)coupled to the conduit (D). The fluid sampling device is configured totransfer a fluid sample from outside the sensor array to the conduit. Anumber of fluid sampling devices may be used including, but not limitedto a syringe needle, a tubing connector, a capillary tube, or a syringeadapter.

[0275] The sensor array may also include a micropump or a microvalvesystem, coupled to the conduit to further aid in the transfer of fluidthrough the conduit. Micropumps and valves have been previouslydescribed. In one embodiment, a micro-valve or micropump may be used tokeep a fluid sample or a reagent solution separated from the sensorarray. Typically, these microvalves and micropumps include a thinflexible diaphragm. The diaphragm may be moved to an open position, inone embodiment, by applying a vacuum to the outside of the diaphragm. Inthis way, a vacuum apparatus coupled to the sensor array may be used toopen a remote microvalve or pump.

[0276] In another embodiment, a microvalve may be used to control theapplication of a vacuum to the system. For example, a microvalve may bepositioned adjacent to the vacuum apparatus. The activation of themicrovalve may allow the vacuum apparatus to communicate with theconduit or sensor array. The microvalve may be remotely activated atcontrolled times and for controlled intervals.

[0277] In one embodiment, a sensor array system, such as depicted inFIG. 39, may be used for analysis of blood samples. A micropuncturedevice (A) is used to extract a small amount of blood from the patient,e.g., through a finger prick. The blood may be drawn through a porousfilter that serves to remove the undesirable particulate matter. For theanalysis of antibodies or antigens in whole blood, the filtering agentmay be chosen to remove both the white and red blood cells, whileleaving in the fluid stream blood plasma and all of the componentstherein. Methods of filtering blood cells from whole blood are taught,for example, in U.S. Pat. Nos. 10 5,914,042; 5,876,605, and 5,211,850which are incorporated by reference. The filtered blood may also bepassed through a reagent delivery reservoir that may consist of a porouslayer that is impregnated with the reagent(s) of interest. In manycases, a visualization agent will be included in this layer so that thepresence of the analytes of interest in the chip can be resolved. Thetreated fluid may be passed above the electronic tongue chip through acapillary layer, down through the various sensing particles and throughthe chip onto the bottom capillary layer. After exiting the centralregion, the excess fluid flows into the vacuum apparatus. This excessfluid may serve as a source of sample for future measurements shouldmore detailed analyses be warranted. A “hard copy” of the sample is thuscreated to back up the electronic data recorded for the specimen.

[0278] Other examples of testing procedures for bodily fluids aredescribed in the following U.S. Pat. Nos. 4,596,657, 4,189,382,4,115,277, 3,954,623, 4,753,776, 4,623,461, 4,069,017, 5,053,197,5,503,985, 3,696,932, 3,701,433, 4,036,946, 5,858,804, 4,050,898,4,477,575, 4,810,378, 5,147,606, 4,246,107, and 4,997,577 all of whichare incorporated by reference.

[0279] This generally described sampling method may also be used foreither antibody or antigen testing of bodily fluids. A general schemefor the testing of antibodies is depicted in FIG. 40. FIG. 40A depicts apolymer bead having a protein coating that can be recognized in aspecific manner by a complimentary antibody. Three antibodies (withinthe dashed rectangle) are shown to be present in a fluid phase thatbathes the polymer bead. Turning to FIG. 40B, the complimentary antibodybinds to the bead while the other two antibodies remain in the fluidphase. A large increase in the complimentary antibody concentration isnoted at this bead. In FIG. 40C a visualization agent such as protein A(within the dashed rectangle) is added to the fluid phase. Thevisualization agent is chosen because it possesses either a strongabsorbance property or it exhibits fluorescence characteristics that canbe used to identify the species of interest via optical measurements.Protein A is an example of a reagent that associates with the commonregion of most antibodies. Chemical derivatization of the visualizationagent with dyes, quantum particles or fluorophores is used to evoke thedesired optical characteristics. After binding to the bead-localizedantibodies, as depicted in FIG. 40D, the visualization agent reveals thepresence of the complimentary antibodies at the specific polymer beadsites.

[0280]FIG. 41 depicts another general scheme for the detection ofantibodies which uses a sensor array composed of four individual beads.Each of the four beads is coated with a different antigen (i.e. aprotein coating). As depicted in FIG. 41A, the beads are washed with afluid sample which includes four antibodies. Each of the four antibodiesbinds to its complimentary antigen coating, as depicted in FIG. 41B. Avisualization agent may be introduced into the chamber, as depicted inFIG. 41C. The visualization agent, in one embodiment, may bind to theantibodies, as depicted in FIG. 41D. The presence of the labeledantibodies is assayed by optical means (absorbance, reflectance,fluorescence). Because the location of the antigen coatings is knownahead of time, the chemicalfbiochemical composition of the fluid phasecan be determined from the pattern of optical signals recorded at eachsite.

[0281] In an alternative methodology, not depicted, the antibodies inthe sample may be exposed to the visualization agent prior to theirintroduction into the chip array. This may render the visualization stepdepicted in 41C unnecessary.

[0282]FIG. 42 depicts a system for detecting an analyte in a fluidstream. The system includes a vacuum apparatus, a chamber in which asensor array may be disposed, and an inlet system for introducing thesample into the chamber. In this embodiment, the inlet system isdepicted as a micro-puncture device. The chamber holding the sensorarray may be a Sikes-Moore chamber, as previously described. The vacuumapparatus is a standard “vacutainer” type vacuum tube. The micropuncture device includes a Luer-lock attachment which can receive asyringe needle. Between the micro-puncture device and the chamber asyringe filter may be placed to filter the sample as the sample entersthe chamber. Alternatively, a reagent may be placed within the filter.The reagent may be carried into the chamber via the fluid as the fluidpasses through the filter.

[0283] As has been previously described, a sensor array may beconfigured to allow the fluid sample to pass through the sensor arrayduring use. The fluid delivery to the sensor array may be accomplishedby having the fluid enter the top of the chip through the showncapillary (A), as depicted in FIG. 43. The fluid flow traverses the chipand exits from the bottom capillary (B). Between the top and bottomcapillaries, the fluid is passed by the bead. Here the fluid containinganalytes have an opportunity to encounter the receptor sites. Thepresence of such analytes may be identified using optical means. Thelight pathway is shown here (D). In the forward flow direction, thebeads are typically forced towards the bottom of the pit. Under thesecircumstances, the bead placement is ideal for optical measurements.

[0284] In another embodiment, the fluid flow may go from the bottom ofthe sensor array toward the top of the sensor array, as depicted in FIG.44. The fluid exits from the top of the chip through the shown capillary(A). The fluid flow traverses the chip and enters from the bottomcapillary (B). Between the top and bottom capillaries, the fluid canavoid the bead somewhat by taking an indirect pathway (C). The presenceof analytes is identified using optical means as before. Unfortunately,only a portion of the light passes through the bead. In the reverse flowdirection, the beads can be dislodged away from the analysis beam by theupwards pressure of the fluid, as shown in FIG. 44. Under thesecircumstances, some of the light may traverse the chip and enter thedetector (not shown) without passing through the sensor bead (Path E).

[0285] In any microfluidic chemical sensing system there may be a needto “store” the chemically sensitive elements in an “inert” environment.Typically, the particles may be at least partially surrounded by aninert fluid such as an inert, non reactive gas, a non-reactive solvent,or a liquid buffer solution. Alternatively, the particles may bemaintained under a vacuum. Before exposure of the particles to theanalyte, the inert environment may need to be removed to allow propertesting of the sample. In one embodiment, a system may include a fluidtransfer system for the removal of an inert fluid prior to theintroduction of the sample with minimum dead volume.

[0286] In one embodiment, a pumping system may be used to pull the inertfluid through from one side (by any pumping action, such as thatprovided by a vacuum downstream from the array). The inert fluid may beefficiently removed while the beads remain within the sensor array.Additionally, the analyte sample may be drawn toward the sensor array asthe inert fluid is removed from the sensor array. A pocket of air mayseparate the analyte sample from the inert fluid as the sample movethrough the conduit. Alternatively, the sample may be pumped from“upstream” using a micropump. Note that a vacuum downstream can producea maximum of one atmosphere of head pressure, while an upstream pumpcould in principle produce an arbitrarily high head pressure. This caneffect the fluid transport rates through the system, but for smallvolume microfluidic systems, even with low flow coefficients, oneatmosphere of head pressure should provide acceptable transfer rates formany applications.

[0287] In another embodiment, the vacuum apparatus may be formeddirectly into a micromachined array. The vacuum apparatus may beconfigured to transmit fluid to and from a single cavity or a pluralityof cavities. In one embodiment, a separate vacuum apparatus may becoupled to each of the cavities.

[0288] 3. Chemical Improvements

[0289] The development of smart sensors capable of discrimination ofdifferent analytes, toxins, and bacteria has become increasinglyimportant for environmental, health and safety, remote sensing,military, and chemical processing applications. Although many sensorscapable of high sensitivity and high selectivity detection have beenfashioned for single analyte detection, only in a few selected caseshave array sensors been prepared which display multi-analyte detectioncapabilities. The obvious advantages of such array systems are theirutility for the analysis of multiple analytes and their ability to be“trained” to respond to new stimuli. Such on site adaptive analysiscapabilities afforded by the array structures makes their utilizationpromising for a variety of future applications.

[0290] Single and multiple analyte sensors both typically rely onchanges in optical signals. These sensors typically make use of anindicator that undergoes a perturbation upon analyte binding. Theindicator may be a chromophore or a fluorophore. A fluorophore is amolecule that absorbs light at a characteristic wavelength and thenre-emits the light most typically at a characteristically differentwavelength. Fluorophores include, but are not limited to rhodamine andrhodamine derivatives, fluorescein and fluorescein derivatives,coumarins and chelators with the lanthanide ion series. The emissionspectra, absorption spectra and chemical composition of manyfluorophores may be found, e.g., in the “Handbook of Fluorescent Probesand Research Chemicals”, R. P. Haugland, ed. which is incorporatedherein by reference. A chromophore is a molecule which absorbs light ata characteristic wavelength, but does not re-emit light.

[0291] As previously described, the receptor itself may incorporate theindicator. The binding of the analyte to the receptor may directly leadto a modulation of the properties of the indicator. Such an approachtypically requires a covalent attachment or strong non-covalent bindingof the indicator onto or as part of the receptor, leading to additionalcovalent architecture. Each and every receptor may need a designedsignaling protocol that is typically unique to that receptor. Generalprotocols for designing in a signal modulation that is versatile andgeneral for most any receptor would be desirable.

[0292] In one embodiment, a general method for the creation of opticalsignal modulations for most any receptor that is coupled to animmobilized matrix has been developed. Immobilized matrices include, butare not limited to, resins, beads, and polymer surfaces. Byimmobilization of the receptor to the matrix, the receptor is heldwithin a structure that can be chemically modified, allowing one to tuneand to create an environment around the receptor that is sensitive toanalyte binding. Coupling of the indicator to an immobilization matrixmay make it sensitive to microenvironment changes which foster signalmodulation of the indicator upon analyte binding. Further, by couplingthe indicator to an immobilization matrix, the matrix itself becomes thesignaling unit, not requiring a specific new signaling protocol for eachand every receptor immobilized on the matrix.

[0293] In an embodiment, a receptor for a particular analyte or class ofanalytes may be designed and created with the chemical handlesappropriate for immobilization on and/or in the matrix. A number of suchreceptors have been described above. The receptors can be, but are notlimited to, antibodies, aptamers, organic receptors, combinatoriallibraries, enzymes, and imprinted polymers.

[0294] Signaling indicator molecules may be created or purchased whichhave appropriate chemical handles for immobilization on and/or in theimmobilization matrix. The indicators may possess chromophores orfluorophores that are sensitive to their microenvironment. Thischromophore or fluorophore may be sensitive to microenvironment changesthat include, but are not limited to, a sensitivity to local pH,solvatophobic or solvatophilic properties, ionic strength, dielectric,ion pairing, and/or hydrogen bonding. Common indicators, dyes, quantumparticles, and semi-conductor particles, are all examples of possibleprobe molecules. The probe molecules may have epitopes similar to theanalyte, so that a strong or weak association of the probe moleculeswith the receptor may occur. Alternatively, the probe molecules may besensitive to a change in their microenvironment that results from one ofthe affects listed in item above.

[0295] Binding of the analyte may do one of the following things,resulting in a signal modulation: 1) displace a probe molecule from thebinding site of the receptor, 2) alter the local pH, 3) change the localdielectric properties, 4) alter the features of the solvent, 5) changethe fluorescence quantum yield of individual dyes, 6) alter therate/efficiency of fluorescence resonance energy transfer (FRET) betweendonor-acceptor fluorophore pairs, or 7) change the hydrogen bonding orion pairing near the probe.

[0296] In an alternative embodiment, two or more indicators may beattached to the matrix. Binding between the receptor and analyte causesa change in the communication between the indicators, again via eitherdisplacement of one or more indicators, or changes in themicroenvironment around one or more indicators. The communicationbetween the indicators may be, but is not limited to, fluorescenceresonance energy transfer, quenching phenomenon, and/or direct binding.

[0297] In an embodiment, a particle for detecting an analyte may becomposed of a polymeric resin. A receptor and an indicator may becoupled to the polymeric resin. The indicator and the receptor may bepositioned on the polymeric resin such that the indicator produces asignal in when the analyte interacts with the receptor. The signal maybe a change in absorbance (for chromophoric indicators) or a change influorescence (for fluorophoric indicators).

[0298] A variety of receptors may be used, in one embodiment, thereceptor may be a polynucleotide, a peptide, an oligosaccharide, anenzyme, a peptide mimetic, or a synthetic receptor.

[0299] In one embodiment, the receptor may be a polynucleotide coupledto a polymeric resin. For the detection of analytes, the polynucleotidemay be a double stranded deoxyribonucleic acid, single strandeddeoxyribonucleic acid, or a ribonucleic acid. Methods for synthesizingand/or attaching a polynucleotide to a polymeric resin are described,for example, in U.S. Pat. No. 5,843,655 which is incorporated herein byreference. “Polynucleotides” are herein defined as chains ofnucleotides. The nucleotides are linked to each other by phosphodiesterbonds. “Deoxyribonucleic acid” is composed of deoxyribonucleotideresidues, while “Ribonucleic acid” is composed of ribonucleotideresidues.

[0300] In another embodiment, the receptor may be a peptide coupled to apolymeric resin. “Peptides” are herein defined as chains of amino acidswhose α-carbons are linked through peptide bonds formed by acondensation reaction between the a carboxyl group of one amino acid andthe amino group of another amino acid. Peptides is intended to includeproteins. Methods for synthesizing and/or attaching a protein orpeptides to a polymeric resin are described, for example, in U.S. Pat.Nos. 5,235,028 and 5,182,366 which is incorporated herein by reference.

[0301] Alternatively, peptide mimetics may be used as the receptor.Peptides and proteins are sequences of amide linked amino acid buildingblocks. A variety of peptide mimetics may be formed by replacing ormodifying the amide bond. In one embodiment, the amide bond may bereplaced by alkene bonds. In another embodiment, the amide may bereplaced by a sulphonamide bond. In another embodiment the amino acidsidechain may be placed on the nitrogen atom, such compounds arecommonly known as peptoids. Peptides may also be formed from non-naturalD-stereo-isomers of amino acids. Methods for synthesizing and/orattaching a peptide mimetic to a polymeric resin is described, forexample, in U.S. Pat. No. 5,965,695 which is incorporated herein byreference.

[0302] In another embodiment, the receptor may include anoligosaccharide coupled to a polymeric resin. An “oligosaccharide” is anoligomer composed of two or more monosaccharides, typically joinedtogether via ether linkages. Methods for synthesizing and/or attachingoligosaccharides to a polymeric resin are described, for example, inU.S. Pat. Nos. 5,278,303 and 5,616,698 which are incorporated herein byreference.

[0303] In another embodiment, polynucleotides, peptides and/oroligosaccharides may be coupled to base unit to form a receptor. In oneembodiment, the base unit may have the general structure:

(R¹)_(n)—X—(R²)_(m)

[0304] wherein X comprises carbocyclic systems or C₁-C₁₀ alkanes, n isan integer of at least 1, m is an integer of at least 1; and

[0305] wherein each of R¹ independently represents—(CH₂)_(y)—NR³—C(NR⁴)—NR⁵, —(CH₂)_(y)—NR⁶R⁷, —(CH₂)_(y)—NH—Y,—(CH₂)_(y)—O-Z;

[0306] where y is an integer of at least 1;

[0307] where R³, R⁴, and R⁵ independently represent hydrogen, alkyl,aryl, alkyl carbonyl of 1 to 10 carbon atoms, or alkoxy carbonyl of 1 to10 carbon atoms, or R⁴ and R⁵ together represent a cycloalkyl group;

[0308] where R⁶ represents hydrogen, alkyl, aryl, alkyl carbonyl of 1 to10 carbon atoms, or alkoxy carbonyl of 1 to 10 carbon atoms;

[0309] where R⁷ represents alkyl, aryl, alkyl carbonyl of 1 to 10 carbonatoms, or alkoxy carbonyl of 1 to 10 carbon atoms;

[0310] where R⁶ and R⁷ together represent a cycloalkyl group;

[0311] where Y is a peptide, or hydrogen

[0312] and where Z is a polynucleotide, an oligosaccharide or hydrogen;and

[0313] wherein each of R² independently represents hydrogen, alkyl,alkenyl, alkynyl, phenyl, 2 phenylalkyl, arylalkyl, aryl, or togetherwith another R² group represent a carbocyclic ring. The use of a baseunit such as described above may aid in the placement and orientation ofthe side groups to create a more effective receptor.

[0314] The receptor and indicators may be coupled to the polymeric resinby a linker group. A variety of linker groups may be used. The term“linker”, as used herein, refers to a molecule that may be used to linka receptor to an indicator; a receptor to a polymeric resin or anotherlinker, or an indicator to a polymeric resin or another linker. A linkeris a hetero or homobifunctional molecule that includes two reactivesites capable of forming a covalent linkage with a receptor, indicator,other linker or polymeric resin. Suitable linkers are well known tothose of skill in the art and include, but are not limited to, straightor branched-chain carbon linkers, heterocyclic carbon linkers, orpeptide linkers. Particularly preferred linkers are capable of formingcovalent bonds to amino groups, carboxyl groups, or sulfhydryl groups orhydroxyl groups. Amino-binding linkers include reactive groups such ascarboxyl groups, isocyanates, isothiocyanates, esters, haloalkyls, andthe like. Carboxyl-binding linkers are capable of forming includereactive groups such as various amines, hydroxyls and the like.Sulfhydryl-binding linkers include reactive groups such as sulfhydrylgroups, acrylates, isothiocyanates, isocyanates and the like. Hydroxylbinding groups include reactive groups such as carboxyl groups,isocyanates, isothiocyanates, esters, haloalkyls, and the like. The useof some such linkers is described in U.S. Pat. No. 6,037,137 which isincorporated herein by reference.

[0315] A number of combinations for the coupling of an indicator and areceptor to a polymeric resin have been devised. These combinations areschematically depicted in FIG. 55. In one embodiment, depicted in FIG.55A, a receptor (R) may be coupled to a polymeric resin. The receptormay be directly formed on the polymeric resin, or be coupled to thepolymeric resin via a linker. An indicator (I) may also be coupled tothe polymeric resin. The indicator may be directly coupled to thepolymeric resin or coupled to the polymeric resin by a linker. In someembodiments, the linker coupling the indicator to the polymeric resin isof sufficient length to allow the indicator to interact with thereceptor in the absence of an analyte.

[0316] In another embodiment, depicted in FIG. 55B, a receptor (R) maybe coupled to a polymeric resin. The receptor may be directly formed onthe polymeric resin, or be coupled to the polymeric resin via a linker.An indicator (B) may also be coupled to the polymeric resin. Theindicator may be directly coupled to the polymeric resin or coupled tothe polymeric resin by a linker. In some embodiments, the linkercoupling the indicator to the polymeric resin is of sufficient length toallow the indicator to interact with the receptor in the absence of ananalyte. An additional indicator (C) may also be coupled to thepolymeric resin. The additional indicator may be directly coupled to thepolymeric resin or coupled to the polymeric resin by a linker. In someembodiments, the additional indicator is coupled to the polymeric resin,such that the additional indicator is proximate the receptor during use.

[0317] In another embodiment, depicted in FIG. 55C, a receptor (R) maybe coupled to a polymeric resin. The receptor may be directly formed onthe polymeric resin, or be coupled to the polymeric resin via a linker.An indicator (I) may be coupled to the receptor. The indicator may bedirectly coupled to the receptor or coupled to the receptor by a linker.In some embodiments, the linker coupling the indicator to the polymericresin is of sufficient length to allow the indicator to interact withthe receptor in the absence of an analyte, as depicted in FIG. 55E.

[0318] In another embodiment, depicted in FIG. 55D, a receptor (R) maybe coupled to a polymeric resin. The receptor may be directly formed onthe polymeric resin, or be coupled to the polymeric resin via a linker.An indicator (B) may be coupled to the receptor. The indicator may bedirectly coupled to the receptor or coupled to the receptor by a linker.In some embodiments, the linker coupling the indicator to the polymericresin is of sufficient length to allow the indicator to interact withthe receptor in the absence of an analyte, as depicted in FIG. 55F. Anadditional indicator (C) may also be coupled to the receptor. Theadditional indicator may be directly coupled to the receptor or coupledto the receptor by a linker.

[0319] In another embodiment, depicted in FIG. 55G, a receptor (R) maybe coupled to a polymeric resin. The receptor may be directly formed onthe polymeric resin, or be coupled to the polymeric resin via a linker.An indicator (B) may be coupled to the polymeric resin. The indicatormay be directly coupled to the polymeric resin or coupled to thepolymeric resin by a linker. In some embodiments, the linker couplingthe indicator to the polymeric resin is of sufficient length to allowthe indicator to interact with the receptor in the absence of ananalyte. An additional indicator (C) may also be coupled to thereceptor. The additional indicator may be directly coupled to thereceptor or coupled to the receptor by a linker.

[0320] In another embodiment, depicted in FIG. 55H, a receptor (R) maybe coupled to a polymeric resin by a first linker. An indicator (I) maybe coupled to the first linker. The indicator may be directly coupled tothe first linker or coupled to the first linker by a second linker. Insome embodiments, the second linker coupling the indicator to thepolymeric resin is of sufficient length to allow the indicator tointeract with the receptor in the absence of an analyte.

[0321] In another embodiment, depicted in FIG. 551, a receptor (R) maybe coupled to a polymeric resin by a first linker. An indicator (B) maybe coupled to the first linker. The indicator may be directly coupled tothe first linker or coupled to the first linker by a second linker. Insome embodiments, the second linker coupling the indicator to the firstlinker is of sufficient length to allow the indicator to interact withthe receptor in the absence of an analyte. An additional indicator (C)may be coupled to the receptor. The additional indicator may be directlycoupled to the receptor or coupled to the receptor by a linker.

[0322] These various combinations of receptors, indicators, linkers andpolymeric resins may be used in a variety of different signallingprotocols. Analyte-receptor interactions may be transduced into signalsthrough one of several mechanisms. In one approach, the receptor sitemay be preloaded with an indicator, which can be displaced in acompetition with analyte ligand. In this case, the resultant signal isobserved as a decrease in a signal produced by the indicator. Thisindicator may be a fluorophore or a chromophore. In the case of afluorophore indicator, the presence of an analyte may be determined by adecrease in the fluorescence of the particle. In the case of achromophore indicator, the presence of an analyte may be determined by adecrease in the absorbance of the particle.

[0323] A second approach that has the potential to provide bettersensitivity and response kinetics is the use of an indicator as amonomer in the combinatorial sequences (such as either structure shownin FIG. 14), and to select for receptors in which the indicatorfunctions in the binding of ligand. Hydrogen bonding or ionicsubstituents on the indicator involved in analyte binding may have thecapacity to change the electron density and/or rigidity of theindicator, thereby changing observable spectroscopic properties such asfluorescence quantum yield, maximum excitation wavelength, maximumemission wavelength, and/or absorbance. This approach may not requirethe dissociation of a preloaded fluorescent ligand (limited in responsetime by k_(off)), and may modulate the signal from essentially zerowithout analyte to large levels in the presence of analyte.

[0324] In one embodiment, the microenvironment at the surface andinterior of the resin beads may be conveniently monitored usingspectroscopy when simple pH sensitive dyes or solvachromic dyes areimbedded in the beads. As a guest binds, the local pH and dielectricconstants of the beads change, and the dyes respond in a predictablefashion. The binding of large analytes with high charge and hydrophobicsurfaces, such as DNA, proteins, and steroids, should induce largechanges in local microenvironment, thus leading to large andreproducible spectral changes. This means that most any receptor can beattached to a resin bead that already has a dye attached, and that thebead becomes a sensor for the particular analyte.

[0325] In one embodiment, a receptor that may be covalently coupled toan indicator. The binding of the analyte may perturb the localmicroenvironment around the receptor leading to a modulation of theabsorbance or fluorescence properties of the sensor.

[0326] In one embodiment, receptors may be used immediately in a sensingmode simply by attaching the receptors to a bead that is alreadyderivatized with a dye sensitive to its microenvironment. This is offersan advantage over other signalling methods because the signalingprotocol becomes routine and does not have to be engineered; only thereceptors need to be engineered. The ability to use several differentdyes with the same receptor, and the ability to have more than one dyeon each bead allows flexibility in the design of a sensing particle.

[0327] Changes in the local pH, local dielectric, or ionic strength,near a fluorophore may result in a signal. A high positive charge in amicroenvironment leads to an increased pH since hydronium migrates awayfrom the positive region. Conversely, local negative charge decreasesthe microenvironment pH. Both changes result in a difference in theprotonation state of pH sensitive indicators present in thatmicroenvironment. Many common chromophores and fluorophores are pHsensitive. The interior of the bead may be acting much like the interiorof a cell, where the indicators should be sensitive to local pH.

[0328] The third optical transduction scheme involves fluorescenceenergy transfer. In this approach, two fluorescent monomers forsignaling may be mixed into a combinatorial split synthesis. Examples ofthese monomers are depicted in FIG. 14. Compound 470 (a derivative offluorescein) contains a common colorimetric/fluorescent probe that maybe mixed into the oligomers as the reagent that will send out amodulated signal upon analyte binding. The modulation may be due toresonance energy transfer to monomer 475 (a derivative of rhodamine).When an analyte binds to the receptor, structural changes in thereceptor will alter the distance between the monomers (schematicallydepicted in FIG. 8, 320 corresponds to monomer 470 and 330 correspondsto monomer 475). It is well known that excitation of fluorescein mayresult in emission from rhodamine when these molecules are orientedcorrectly. The efficiency of resonance energy transfer from fluoresceinto rhodamine will depend strongly upon the presence of analyte binding;thus measurement of rhodamine fluorescence intensity (at a substantiallylonger wavelength than fluorescein fluorescence) will serve as aindicator of analyte binding. To greatly improve the likelihood of amodulatory fluorescein-rhodamine interaction, multiple rhodamine tagscan be attached at different sites along a combinatorial chain withoutsubstantially increasing background rhodamine fluorescence (onlyrhodamine very close to fluorescein will yield appreciable signal). Inone embodiment, depicted in FIG. 8, when no ligand is present, shortwavelength excitation light (blue light) excites the fluorophore 320,which fluoresces (green light). After binding of analyte ligand to thereceptor, a structural change in the receptor molecule bringsfluorophore 320 and fluorophore 330 in proximity, allowing excited-statefluorophore 320 to transfer its energy to fluorophore 330. This process,fluorescence resonance energy transfer, is extremely sensitive to smallchanges in the distance between dye molecules (e.g.,efficiency˜[distance]⁻⁶).

[0329] In another embodiment, photoinduced electron transfer (PET) maybe used to analyze the local microenvironment around the receptor. Themethods generally includes a fluorescent dye and a fluorescencequencher. A fluorescence quencher is a molecule that absorbs the emittedradiation from a fluorescent molecule. The fluorescent dye, in itsexcited state, will typically absorbs light at a characteristicwavelength and then re-emit the light at a characteristically differentwavelength. The emitted light, however, may be reduced by electrontransfer with the fluorescent quncher, which results in quenching of thefluorescence. Therefore, if the presence of an analyte perturbs thequenching properties of the fluorescence quencher, a modulation of thefluorescent dye may be observed.

[0330] The above described signalling methods may be incorporated into avariety of receptor-indicator-polymeric resin systems. Turning to FIG.55A, an indicator (I) and receptor (R) may be coupled to a polymericresin. In the absence of an analyte, the indicator may produce a signalin accordance with the local microenvironment. The signal may be anabsorbance at a specific wavelength or a fluorescence. When the receptorinteracts with an analyte, the local microenvironment may be alteredsuch that the produced signal is altered. In one embodiment, depicted inFIG. 55A, the indicator may partially bind to the receptor in theabsence of an analyte. When the analyte is present the indicator may bedisplaced from the receptor by the analyte. The local microenvironmentfor the indicator therefore changes from an environment where theindicator is binding with the receptor, to an environment where theindicator is no longer bound to the receptor. Such a change inenvironment may induce a change in the absorbance or fluorescence of theindicator.

[0331] In another embodiment, depicted in Turning to FIG. 55C, anindicator (I) may be coupled to a receptor (R). The receptor may becoupled to a polymeric resin. In the absence of an analyte, theindicator may produce a signal in accordance with the localmicroenvironment. The signal may be an absorbance at a specificwavelength or a fluorescence. When the receptor interacts with ananalyte, the local microenvironment may be altered such that theproduced signal is altered. In contrast to the case depicted in FIG.55A, the change in local microenvironment may be due to a conformationchange of the receptor due to the biding of the analyte. Such a changein environment may induce a change in the absorbance or fluorescence ofthe indicator.

[0332] In another embodiment, depicted in FIG. 55E, an indicator (I) maybe coupled to a receptor by a linker. The linker may have a sufficientlength to allow the indicator to bind to the receptor in the absence ofan analyte. The receptor (R) may be coupled to a polymeric resin. In theabsence of an analyte, the indicator may produce a signal in accordancewith the local microenvironment. As depicted in FIG. 55E, the indicatormay partially bind to the receptor in the absence of an analyte. Whenthe analyte is present the indicator may be displaced from the receptorby the analyte. The local microenvironment for the indicator thereforechanges from an environment where the indicator is binding with thereceptor, to an environment where the indicator is no longer bound tothe receptor. Such a change in environment may induce a change in theabsorbance or fluorescence of the indicator.

[0333] In another embodiment, depicted in FIG. 55H, a receptor (R) maybe coupled to a polymeric resin by a first linker. An indicator may becoupled to the first linker. In the absence of an analyte, the indicatormay produce a signal in accordance with the local microenvironment.

[0334] The signal may be an absorbance at a specific wavelength or afluorescence. When the receptor interacts with an analyte, the localmicroenvironment may be altered such that the produced signal isaltered. In one embodiment, as depicted in FIG. 55H, the indicator maypartially bind to the receptor in the absence of an analyte. When theanalyte is present the indicator may be displaced from the receptor bythe analyte. The local microenvironment for the indicator thereforechanges from an environment where the indicator is binding with thereceptor, to an environment where the indicator is no longer bound tothe receptor. Such a change in environment may induce a change in theabsorbance or fluorescence of the indicator.

[0335] In another embodiment, the use of fluorescence resonance energytransfer or photoinduced electron transfer may be used to detect thepresence of an analyte. Both of these methodologies involve the use oftwo fluorescent molecules. Turning to FIG. 55B, a first fluorescentindicator (B) may be coupled to receptor (R). Receptor (R) may becoupled to a polymeric resin. A second fluorescent indicator (C) mayalso be coupled to the polymeric resin. In the absence of an analyte,the first and second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. In one embodiment, excitation ofthe first fluorescent indicator may result in emission from the secondfluorescent indicator when these molecules are oriented correctly.Alternatively, either the first or second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, the presenceof an analyte may cause the indicators to move further apart. This hasan effect of reducing the fluorescent energy transfer. If the twoindicators interact to produce an emission signal in the absence of ananalyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching and thefluorescent emission from the other indicator may increase. It should beunderstood that these effects will reverse if the presence of an analytecauses the indicators to move closer to each other.

[0336] In another embodiment, depicted in FIG. 55D, a first fluorescentindicator (B) may be coupled to receptor (R). A second fluorescentindicator (C) may also be coupled to the receptor. Receptor (R) may becoupled to a polymeric resin. In the absence of an analyte, the firstand second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. In one embodiment, excitation ofthe first fluorescent indicator may result in emission from the secondfluorescent indicator when these molecules are oriented correctly.Alternatively, either the first or second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, depicted inFIG. 55D, the presence of an analyte may cause the indicators to movefurther apart. This has an effect of reducing the fluorescent energytransfer. If the two indicators interact to produce an emission signalin the absence of an analyte, the presence of the analyte may cause adecrease in the emission signal. Alternatively, if one the indicators isa fluorescence quencher, the presence of an analyte may disrupt thequenching and the fluorescent emission from the other indicator mayincrease. It should be understood that these effects will reverse if thepresence of an analyte causes the indicators to move closer to eachother.

[0337] In a similar embodiment to FIG. 55D, the first fluorescentindicator (B) and second fluorescent indicator (C) may be both coupledto receptor (R), as depicted in FIG. 55F. Receptor (R) may be coupled toa polymeric resin. First fluorescent indicator (B) may be coupled toreceptor (R) by a linker group. The linker group may allow the firstindicator to bind the receptor, as depicted in FIG. 55F. In the absenceof an analyte, the first and second fluorescent indicators may bepositioned such that fluorescence energy transfer may occur. When theanalyte is presence, the first indicator may be displaced from thereceptor, causing the fluorescence energy transfer between the twoindicators to be altered.

[0338] In another embodiment, depicted in FIG. 55G, a first fluorescentindicator (B) may be coupled to a polymeric resin. Receptor (R) may alsobe coupled to a polymeric resin. A second fluorescent indicator (C) maybe coupled to the receptor (R). In the absence of an analyte, the firstand second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. In one embodiment, excitation ofthe first fluorescent indicator may result in emission from the secondfluorescent indicator when these molecules are oriented correctly.Alternatively, either the first or second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, the presenceof an analyte may cause the indicators to move further apart. This hasan effect of reducing the fluorescent energy transfer. If the twoindicators interact to produce an emission signal in the absence of ananalyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching and thefluorescent emission from the other indicator may increase. It should beunderstood that these effects will reverse if the presence of an analytecauses the indicators to move closer to each other.

[0339] In another embodiment, depicted in FIG. 55I, a receptor (R) maybe coupled to a polymeric resin by a first linker. A first fluorescentindicator (B) may be coupled to the first linker. A second fluorescentindicator (C) may be coupled to the receptor (R). In the absence of ananalyte, the first and second fluorescent indicators may be positionedsuch that fluorescence energy transfer may occur. In one embodiment,excitation of the first fluorescent indicator may result in emissionfrom the second fluorescent indicator when these molecules are orientedcorrectly. Alternatively, either the first or second fluorescentindicator may be a fluorescence quencher. When the two indicators areproperly aligned, the excitation of the fluorescent indicators mayresult in very little emission due to quenching of the emitted light bythe fluorescence quencher. In both cases, the receptor and indicatorsmay be positioned such that fluorescent energy transfer may occur in theabsence of an analyte. When the analyte is presence the orientation ofthe two indicators may be altered such that the fluorescence energytransfer between the two indicators is altered. In one embodiment, thepresence of an analyte may cause the indicators to move further apart.This has an effect of reducing the fluorescent energy transfer. If thetwo indicators interact to produce an emission signal in the absence ofan analyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching and thefluorescent emission from the other indicator may increase. It should beunderstood that these effects will reverse if the presence of an analytecauses the indicators to move closer to each other.

[0340] In one embodiment, polystyrene/polyethylene glycol resin beadsmay be used as a polymeric resin since they are highly water permeable,and give fast response times to penetration by analytes. The beads maybe obtained in sizes ranging from 5 microns to 250 microns. Analysiswith a confocal microscope reveals that these beads are segregated intopolystyrene and polyethylene glycol microdomains, at about a 1 to 1ratio. Using the volume of the beads and the reported loading of 300pmol/bead, we can calculate an average distance of 35 Å between terminalsites. This distance is well within the Forester radii for thefluorescent dyes that we are proposing to use in our fluorescenceresonance energy transfer (“FRET”) based signaling approaches. Thisdistance is also reasonable for communication between binding events andmicroenvironment changes around the fluorophores.

[0341] The derivatization of the beads with our receptors and with theindicators may be accomplished by coupling carboxylic acids and aminesusing EDC and HOBT. Typically, the efficiency of couplings are greaterthat 90% using quantitative ninhydrin tests. (See Niikura, K.; Metzger,A.; and Anslyn, E. V. “A Sensing Ensemble with Selectivity for IositolTrisphosphate”, J. Am. Chem. Soc. 1998, 120, 0000, which is incorporatedherein by reference). The level of derivatization of the beads issufficient to allow the loading of a high enough level of indicators andreceptors to yield successful assays. However, an even higher level ofloading may be advantageous since it would increase the multi-valencyeffect for binding analytes within the interior of the beads. We mayincrease the loading level two fold and ensure that two amines are closein proximity by attaching an equivalent of lysine to the beads (see FIG.45D).

[0342] The amines may be kept in proximity so that binding of an analyteto the receptor will influence the environment of a proximal indicator.

[0343] Even though a completely random attachment of indicator and areceptor lead to an effective sensing particle, it may be better torationally place the indicator and receptor in proximity. In oneembodiment, lysine that has different protecting groups on the twodifferent amines may be used, allowing the sequential attachment of anindicator and a receptor. If needed, additional rounds of derivatizationof the beads with lysine may increase the loading by powers of two,similar to the synthesis of the first few generations of dendrimers.

[0344] In contrast, too high a loading of fluorophores will lead toself-quenching, and the emission signals may actually decrease withhigher loadings. If self quenching occurs for fluorophores on thecommercially available beads, we may incrementally cap the terminalamines thereby giving incrementally lower loading of the indicators.

[0345] Moreover, there should be an optimum ratio of receptors toindicators. The optimum ratio is defined as the ratio of indicator toreceptor to give the highest response level. Too few indicators comparedto receptors may lead to little change in spectroscopy since there willbe many receptors that are not in proximity to indicators. Too manyindicators relative to receptors may also lead to little change inspectroscopy since many of the indicators will not be near receptors,and hence a large number of the indicators will not experience a changein microenvironment. Through iterative testing, the optimum ratio may bedetermined for any receptor indicator system.

[0346] This iterative sequence will be discussed in detail for aparticle designed to signal the presence of an analyte in a fluid. Thesequence begins with the synthesis of several beads with differentloadings of the receptor. The loading of any receptor may be quantitatedusing the ninhydrin test. (The ninhydrin test is described in detail inKaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. “Color Testfor Detection of Free Terminal Amino Groups in the Solid-Phase Synthesisof Peptides”, Anal. Biochem. 1970, 34, 595-598 which is incorporatedherein by reference). The number of free amines on the bead is measuredprior to and after derivatization with the receptor, the difference ofwhich gives the loading. Next, the beads undergo a similar analysis withvarying levels of molecular probes. The indicator loading may bequantitated by taking the absorption spectra of the beads. In thismanner, the absolute loading level and the ratio between the receptorand indicators may be adjusted. Creating calibration curves for theanalyte using the different beads will allow the optimum ratios to bedetermined.

[0347] The indicator loading may be quantitated by taking the absorptionspectra of a monolayer of the beads using our sandwich technique (SeeFIG. 46D). The sandwich technique involves measuring the spectroscopy ofsingle monolayers of the beads. The beads may be sandwiched between twocover slips and gently rubbed together until a monolayer of the beads isformed. One cover slip is removed, and mesh with dimensions on the orderof the beads is then place over the beads, and the cover slip replaced.This sandwich is then placed within a cuvette, and the absorbance oremission spectra are recorded. Alternatively, an sensor array system, asdescribed above, may be used to analyze the interaction of the beadswith the analyte.

[0348] A variety of receptors may be coupled to the polymeric beads.Many of these receptors have been previously described. Other receptorsare shown in FIG. 47.

[0349] As described generally above, an ensemble may be formed by asynthetic receptor and a probe molecule, either mixed together insolution or bound together on a resin bead. The modulation of thespectroscopic properties of the probe molecule results from perturbationof the microenvironment of the probe due to interaction of the receptorwith the analyte; often a simple pH effect. The use of a probe moleculecoupled to a common polymeric support may produce systems that givecolor changes upon analyte binding. A large number of dyes arecommercially available, many of which may be attached to the bead via asimple EDC/HOBT coupling (FIG. 48 shows some examples of indicators).These indicators are sensitive to pH, and also respond to ionic strengthand solvent properties. When contacted with an analyte, the receptorinteracts with the analyte such that microenvironment of the polymericresin may become significantly changed. This change in themicroenvironment may induce a color change in the probe molecule. Thismay lead to an overall change in the appearance of the particleindicating the presence of the analyte.

[0350] Since many indicators are sensitive to pH and local ionicstrength, index of refraction, and/or metal binding, lowering the localdielectric constant near the indicators may modulate the activity of theindicators such that they are more responsive. A high positive charge ina microenvironment leads to an increased pH since hydronium ions migrateaway from the positive region. Conversely, local negative chargedecreases the microenvironment pH. Both changes result in a differenceon the protonation state of a pH sensitive indicator present in thatmicroenvironment. The altering of the local dielectric environment maybe produced by attaching molecules of differing dielectric constants tothe bead proximate to the probe molecules. Examples of molecules whichmay be used to alter the local dielectric environment include, but arenot limited to, planar aromatics, long chain fatty acids, and oligomerictracts of phenylalanine, tyrosine, and tryptophan. Differing percentagesof these compounds may be attached to the polymeric bead to alter thelocal dielectric constant.

[0351] Competition assays may also be used to produce a signal toindicate the presence of an analyte. The high specificity of antibodiesmakes them the current tools of choice for the sensing and quantitationof structurally complex molecules in a mixture of analytes. These assaysrely on a competition approach in which the analyte is tagged and boundto the antibody. Addition of the untagged analyte results in a releaseof the tagged analytes and spectroscopic modulation is monitored.Surprisingly, although competition assays have been routinely used todetermine binding constants with synthetic receptors, very little workhas been done exploiting competition methods for the development ofsensors based upon synthetic receptors. Yet, all the ways in which themicroenvironment of the chromophore can be altered, as described above,may be amenable to the competition approach. Those that have beendeveloped using synthetic receptors are mostly centered upon the use ofcyclodextrins. (See e.g., Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno,A.; Toda, F.; Suzuki, I.; Osa, T. “Fluorescent Sensors of MolecularRecognition. Modified Cyclodextrins Capable of ExhibitingGuest-Responsive Twisted Intramolecular Charge Transfer Fluorescence” J.Am. Chem. Soc. 1993, 115, 5035, and reference (5) therein, which areincorporated herein by reference) A series of parent and derivatizedcyclodextrins have been combined with chromophores that are responsiveto the hydrophobicity of their microenvironment to produce a sensorsystem. Displacement of the chromophores from the cyclodextrin cavity bybinding of a guest leads to a diagnostic spectroscopy change.

[0352] This competitive approach has been used successfully, in oneembodiment, for the detection of carbohydrates such asinositol-1,4,5-triphosphate (IP₃). In one embodiment, a syntheticreceptor 5 may be paired with an optical signaling molecule5-carboxyfluorescein, to quantitate IP₃ at nM concentrations. Acompetition assay employing an ensemble of 5-carboxyfluorescein andreceptor 5 was used to measure binding constants. The addition ofreceptor 5 to 5-carboxyfluorescein resulted in a red shift of theabsorption of 5-carboxyfluorescein. Monitoring the absorption at 502 nm,followed by analysis of the data using the Benesi-Hildebrand method,gave affinity constants of 2.2×10⁴ M⁻¹ for 5-carboxyfluorescein bindingto receptor 5. Addition of IP₃ to a solution of the complexes formedbetween 5 and 5-carboxyfluorescein resulted in displacement of5-carboxyfluorescein and a subsequent blue shift.

[0353] In order to enhance the affinity of receptor 5 for IP₃, similarassays were repeated in methanol, and with 2% of the surfactantTriton-X. In methanol and the detergent solutions, 5-carboxyfluoresceinprefers a cyclized form in which the 2-carboxylate has undergone anintramolecular conjugate addition to the quinoid structure. This form of5-carboxyfluorescein is colorless and nonfluorescent. Upon addition ofreceptor 5 the yellow color reappears as does the fluorescence. Thepositive character of the receptor induces a ring opening to give thecolored/fluorescent form of 5-carboxyfluorescein. Using theBenesi-Hildebrand method applied to absorption data a binding constantof 1.2×10⁵ M⁻¹ was found for receptor 5 and 5-carboxyfluorescein. Asanticipated based upon the differences in the spectroscopy of5-carboxyfluorescein when it is bound to receptor 5 or free in solution,addition of IP₃ to a solution of receptor 5 and 5-carboxyfluoresceinresulted in a decrease of absorbance and fluorescence due to release of5-carboxyfluorescein into the methanol solution. Binding constants of1.0×10⁸ M⁻¹ and 1.2×10⁷ M⁻¹ for IP₃ and receptor 5 were found formethanol and the surfactant solution respectively.

[0354] Since fluorescence spectroscopy is a much more sensitivetechnique than UV/visible spectroscopy, and the use of methanol gavesignificantly stronger binding between receptor 5 and5-carboxyfluorescein, as well as between receptor 5 and IP₃, themonitoring of fluorescence was found to be the method of choice forsensing nM concentrations of IP₃. We find that the addition of IP₃ to anensemble of receptor 5 and 5-carboxyfluorescein in water may detect andquantitate IP₃ at a concentration as low as 1 mM. Importantly, inmethanol a 10 nM IP₃ concentration was easily detected. A detectionlevel in the nM range is appropriate for the development of an assayusing methanol or surfactant as an eluent and capillary electrophoresisto sample and fractionate cellular components.

[0355] We have shown that receptor 5 binds IP₃ quite selectively overother similarly charged species present in cells. Polyanions withcharges higher than IP₃, such as IP₄, IP₅, and oligonucleotides,however, are expected to bind with higher affinities. In order tofractionate the cellular components during signal transduction, andspecifically monitor IP₃, a combination of a chemically sensitiveparticle and capillary electrophoresis (CE) may be used. As has beendescribed above, a sensor array may include a well in which the particleis placed, along with a groove in which the capillary will reside. Thecapillary will terminate directly into the interior of the bead (SeeFIG. 49). Illumination from above and CCD analysis from below may beused to analyze the particle. Samples as small as 100 femtoliters may beintroduced into an electrophoresis capillary for analysis. Using highsensitivity multiphoton-excited fluorescence as few as ˜50,000 moleculesof various precursors/metabolites of the neurotransmitter, serotonin maybe detected. Cytosolic samples may be collected and fractionated inmicron-diameter capillary electrophoresis channels. At the capillaryoutlet, components may migrate from the channel individually, and willbe directed onto a bead that houses immobilized receptor 5 and the dyesappropriate for our various signaling strategies. Receptor binding ofIP₃ or IP₄ will elicit modulations in the emission and/or absorptionproperties.

[0356] Dramatic spectroscopy changes accompany the chelation of metalsto ligands that have chromophores. In fact, mostcolorimetric/fluorescent sensors for metals rely upon such a strategy.Binding of the metal to the inner sphere of the ligand leads toligand/metal charge transfer bands in the absorbance spectra, andchanges in the HOMO-LUMO gap that leads to fluorescence modulations.

[0357] In one embodiment, the binding of an analyte may be coupled withthe binding of a metal to a chromophoric ligand, such that the metal maybe used to trigger the response of the sensor for the analyte. Thecompound known as Indo-1 (see FIG. 50 for the structure and emissionproperties) is a highly fluorescent indicator that undergoes a largewavelength shift upon exposure to Ca(II). Further, compound 2 bindsCe(III) and the resulting complex is fluorescent. In one embodiment, thebinding of Ca(II) or Ce(III) to these sensors may be altered by theaddition of an analyte of interest. By altering the binding of thesemetals to a receptor a signal may be generated indicating the presenceof the receptor.

[0358] In one embodiment, fluorescent indicators that have been used tomonitor Ca(II) and Ce(III) levels in other applications may be appliedto a polymeric supported system. Using the Ca(II) sensor Indo-1 as anexample, the strategy is shown in FIG. 51. Indo-1 binds Ca(II) at nMconcentrations (see FIG. 50). Attachment of Indo-1 and one of ourguanidinium/amine based receptors 3-6 to a resin bead (derivatized withlysine as depicted in FIG. 45D) may lead to intramolecular interactionsbetween the carboxylates of Indo-1 and the guanidiniums/ammoniums of areceptor. The coordination of the carboxylates of Indo-1 may result in adecreased affinity for Ca(II). However, there should be cooperativebinding of Ca(II) and our analytes. Once one of the anionic analytes isbound to its respective receptor, it will competitively displace thecarboxylates of Indo-1 leading to increased Ca(II) binding, which inturn will result in a fluorescence modulation. Similarly, binding ofCa(II) to Indo-1 leaves the guanidiniums of the receptors free to bindcitrate. The assays will likely be most sensitive at concentrations ofthe analytes and Ca(II) near their dissociation constants, where neitherreceptor is saturated and small changes in the extent of binding lead tolarge changes in fluorescence.

[0359] We also may switch the role of the metal and the ligand. Indo-1is fluorescent with and without the Ca(II). However, compound 2 is notfluorescent until Ce(III) binds to it. Thus, a similar assay that reliesupon a change of microenvironment in the interior of the bead dependingupon the presence or absence of the analyte should perturb the bindingof Ce(III) to compound 2. In this case, a repulsive interaction ispredicted for the binding of Ce(III) when the positive charges of theguanidinium based receptors are not neutralized by binding to theanionic analytes.

[0360] In one embodiment, an indicator may be coupled to a bead andfurther may be bound to a receptor that is also coupled to the bead.Displacement of the indicator by an analyte will lead to signalmodulation. Such a system may also take advantage of fluorescentresonance energy transfer to produce a signal in the presence of ananalyte. Fluorescence resonance energy transfer is a technique that canbe used to shift the wavelength of emission from one position to anotherin a fluorescence spectra. In this manner it creates a much moresensitive assay since one can monitor intensity at two wavelengths. Themethod involves the radiationless transfer of excitation energy from onefluorophore to another. The transfer occurs via coupling of theoscillating dipoles of the donor with the transition dipole of theacceptor. The efficiency of the transfer is described by equations firstderived by Forester. They involve a distance factor (R), orientationfactor (k), solvent index of refraction (N), and spectral overlap (J).

[0361] In order to incorporate fluorescence resonance energy transferinto a particle a receptor and two different indicators may beincorporated onto a polymeric bead. In the absence of an analyte thefluorescence resonance energy transfer may occur giving rise to adetectable signal. When an analyte interacts with a receptor, thespacing between the indicators may be altered. Altering this spacing maycause a change in the fluorescence resonance energy transfer, and thus,a change in the intensity or wavelength of the signal produced. Thefluorescence resonance energy transfer efficiency is proportional to thedistance (R) between the two indicators by 1/R⁶. Thus slight changes inthe distance between the two indicators may induce significant changesin the fluorescence resonance energy transfer.

[0362] In one embodiment, various levels of coumarin and fluorescein maybe loaded onto resin beads so as to achieve gradiations in FRET levelsfrom zero to 100%. FIG. 52 shows a 70/30 ratio of emission from5-carboxyfluorescein and coumarin upon excitation of coumarin only inwater. However, other solvents give dramatically different extents ofFRET. This shows that the changes in the interior of the beads does leadto a spectroscopic response. This data also shows that differentialassociation of the various solvents and 5-carboxyfluorescein on resinbeads as a function of solvents. This behavior is evoked from thesolvent association with the polymer itself, in the absence ofpurposefully added receptors. We may also add receptors which exhibitstrong/selective association with strategic analytes. Such receptors mayinduce a modulation in the ratio of FRET upon analyte binding, withinthe microenvironment of the polystyrene/polyethylene glycol matrices.

[0363] In order to incorporate a wavelength shift into a fluorescenceassays, receptors 3-6 may be coupled to thecourmarin/5-carboxyfluorescein beads discussed above. When5-carboxyfluorescein is bound to the various receptors and coumarin isexcited, the emission will be primarily form coumarin since thefluorescein will be bound to the receptors. Upon displacement of the5-carboxyfluorescein by the analytes, emission should shift more toward5-carboxyfluorescein since it will be released to the bead environmentwhich possesses coumarin. This will give us a wavelength shift in thefluorescence which is inherently more sensitive than the modulation ofintensity at a signal wavelength.

[0364] There should be large changes in the distance between indicators(R) on the resin beads. When the 5-carboxyfluorescein is bound, thedonor/acceptor pair should be farther than when displacement takesplace; the FRET efficiency scales as 1/R⁶. The coumarin may be coupledto the beads via a floppy linker, allowing it to adopt manyconformations with respect to a bound 5-carboxyfluorescein. Hence, it ishighly unlikely that the transition dipoles of the donor and acceptorwill be rigorously orthogonal.

[0365] In one embodiment, a receptor for polycarboxylic acids and anappropriate probe molecule may be coupled to a polymeric resin to form aparticle for the detection of polycarboxylic acid molecules. Receptorsfor polycarboxylic acids, as well as methods for their use in thedetection of polycarboxylic acids, have been described in U.S. Pat. No.6,045,579 which is incorporated herein by reference. This systeminvolves, in one embodiment, the use of a receptor 3 which was found tobe selective for the recognition of a tricarboxylic acid (e.g., citrate)in water over dicarboxylates, monocarboxylates, phosphates, sugars, andsimple salts. The receptor includes guanidinium groups for hydrogenbonding and charge pairing with the tricarboxylic acid.

[0366] An assay for citrate has employed an ensemble of5-carboxyfluorescein and 3. The binding between 3 and5-carboxyfluorescein resulted in a lowering of the phenol pK_(a) of5-carboxyfluorescein, due to the positive microenvironment presented by3. This shift in pK_(a) (local pH) caused the phenol moiety to be in ahigher state of protonation when 5-carboxyfluorescein was free insolution. The absorbance or fluorescence of 5-carboxyfluoresceindecreases with higher protonation of the phenol. The intensity ofabsorbance increases with addition of host 3 to 5-carboxyfluorescein,and as predicted the intensity decreases upon addition of citrate to theensemble of 3 and 5-carboxyfluorescein. The same effect was seen in thefluorescence spectrum (λmax=525 nm).

[0367] In an embodiment, a metal may be used to trigger the response ofa chromophore to the presence of an analyte. For example, compound 7binds Cu(II) with a binding constant of 4.9×10⁵ M⁻¹ (See FIG. 53).Addition of 1 eq. of Cu(II) increases the binding constant of citrate tocompound 7 by a factor of at least 5. Importantly, the addition ofcitrate increases the binding of Cu(II) to the receptor by a factor ofat least 10. Therefore the citrate and Cu(II) enhance each other'sbinding in a cooperative manner. Further, the emission spectra ofcompound 7 is quite sensitive to the addition of citrate when Cu(II) ispresent, but has no response to the addition of citrate in the absenceof Cu(II). Thus the binding of a “trigger” may be perturbed with ananalyte of interest, and the perturbation of the binding of the triggermay be used to spectroscopically monitor the binding of the analyte. Thetriggering of the sensing event by an added entity is similar to therequirement for enzymes in saliva to degrade food particulants intotastants recognizable by the receptors on mammalian taste buds.

[0368] In one embodiment, citrate receptor 3 may be immobilized on apolystyrene/polyethylene glycol bead, where on the same bead may also beattached a fluorescent probe molecule (FIG. 54). Solutions of citrate atdifferent concentrations may be added to the beads, and the fluorescencespectra of the monolayer recorded. We find exactly the same fluorescenceresponse toward citrate for the ensemble of receptor 3 and5-carboxyfluorescein on the beads as in solution. Apparently, a similarmicroenvironment change to modulate the spectroscopy of5-carboxyfluorescein occurs in the beads, although both5-carboxyfluorescein and receptor 3 are just randomly placed throughoutthe bead.

[0369] Additional sensor system include sensors for tartrate andtetracyclin. Compound 4 binds tartrate in buffered water (pH 7.4) with abinding constant of approximately 10⁵ M⁻¹. The binding is slow on theNMR time scale, since we can observe both the bound and free receptorand tartrate. This binding is surprisingly strong for pure water. Itmust reflect good cooperativity between the host's boronic acid moietyand the two guanidinium groups for the recognition of the guest'svicinal diol and two carboxylates respectively. Compound 6 may act as amolecular receptor for tetracyclin. The compound has been synthesized,and by variable temperature NMR it has been found to be in a bowlconformation. Its binding properties with several indicators have beenexplored (most bind with affinities near 10⁴ M⁻¹). More importantly, thebinding of tetracyclin has also been explored, and our preliminaryresults suggests that the binding constant in water is above 10³ M⁻¹.

[0370] In another embodiment, a sensing particle may include an oligomerof amino acids with positively charged side chains such as the lysinetrimer, depicted in FIG. 56, designed to act as the anion receptor, andan attached FRET pair for signaling. Sensing of different anions may beaccomplished by optically monitoring intensity changes in the signal ofthe FRET pair as the analyte interacts with the oligomer.

[0371] Upon introduction of an anionic species to 1, the analyte maybind to the trimer, disturbing the trimer-fluorescein interaction,thereby, altering the fluorescein's ability to participate in the energytransfer mechanism. Using a monolayer of resin in a conventionalfluorometer, the ratio of D:A emission for the FRET pair attached toTG-NH₂ resin is sensitive to different solvents as well as to the ionicstrength of the solution. Epifluorescence studies may be performed totest the solvent dependence, ionic strength, and binding effects ofdifferent anions on the FRET TG-NH₂ resins. The images of the FRETTG-NH₂ resins within a sensor array, taken by a charged coupled device(CCD) may result in three output channels of red, green, and blue lightintensities. The RGB light intensities will allow for comparison of theresults obtained using a conventional fluorometer.

[0372] The signal transduction of 1 may be studied using a standardfluorometer and within the array platform using epifluorescencemicroscopy. The RGB analysis may be used to characterize the relativechanges in emission of the FRET pair. Other resin-bound sensors may besynthesized by varying the amino acid subunits within the oligomers andthe length of the peptide chains.

[0373] In another embodiment, solvatochromic dyes may be covalentlylinked to a receptor unit tethered to a resin bead that is capable ofbinding to small organic guests. In one example, dansyl and dapoxyl mayact as sensitive probes of their microenvironment. When selecting a dyefor use, characteristics such as high extinction coefficients, highfluorescence quantum yields, and large Stoke's shifts should beconsidered. Dapoxyl and dansyl were anchored to 6% agarose resin beads,in an effort to enhance the signaling response of these resin boundfluorophores in various solvent systems. Agarose beads are crosslinkedgalactose polymers that are more hydrophilic than thepolystyrene-polyethylene glycol resins. The attachment of thesesolvatochromic dyes to the agarose resin beads is outlined in FIG. 57.

[0374] The dapoxyl labeled resin (6) was formed by reductively aminatingglyoxalated agarose resin with mono (Fmoc)-butyldiamine hydrochloridesalt using sodium borohydride as the reducing agent. The base labileprotecting group, Fmoc, was removed from 3 with dilute base, and thesolvatochromic dye was anchored to 4 through a reaction to form asulfonamide bond resulting in 6. The tethering of dansyl to agaroseresin was performed similarly.

[0375] Analysis of the agarose resins derivatized with dansyl anddapoxyl was attempted several times using a monolayer sample cell in aconventional fluorometer. However, satisfactory emission spectra of 5and 6 in different solvent systems were not obtained due to the fragilenature of the agarose resin which placed restrictions on themanufacturing of the monolayer sample cell.

[0376] Significant signal enhancement of 5 and 6 was seen when thesolvent system was changed from a 50 mM phosphate buffer (pH=7.0) toethanol (EtOH), methanol (MeOH), and acetonitrile (CH₃CN). The emissionof 6 increased three fold in EtOH and five fold in CH₃CN when comparedto the emission of 6 in a buffer. The agarose-dansyl resin, 5,demonstrated similar trends in response to different solvents; however,the intensities were smaller than for 6. For instance, the emission of 5in EtOH for the red channel was 61% smaller in intensity units comparedto 6 (2200 vs. 5800 arbitrary intensity units). This observation hasbeen attributed to the lower quantum yield of fluorescence and thesmaller extinction coefficient of dansyl to that of dapoxyl. From theseinitial studies, the average fluorescence intensity of the three beadsof type 6 in EtOH across the red channel was 5800±300 arbitraryintensity counts with a percent standard deviation of 5.0 %. Also,before changing to a new solvent, the agarose beads were flushed withthe buffer for 5 minutes in order to return the agarose-dye resin to a“zero” point of reference. The background variance of the fluorescenceintensity of 6 when exposed to each of the buffer washes between eachsolvent system was 5.0% and 4.0% in the red and green channels,respectively.

[0377] The response of 5 and 6 to varying ratios of two differentsolvents was also studied. As seen in FIG. 58, a detectable decrease inthe emission of 6 is observed as the percent of the 50 mM phosphatebuffer (pH=7) is increased in ethanol. The fluorescence intensity of 6decreased by three fold from its original value in 100% EtOH to 100%buffer. There was an incremental decrease in the fluorescence emissionintensities of 6 in both the red and green channels. Once again, 5demonstrated similar trends in response to the varying ratios of mixedsolvent systems; however, the intensities were smaller than 6.

[0378] In another example, each dye was derivatized with benzyl amine(2-4) for studies in solution phase and anchored to resin (5-7) forstudies using the sandwich method and epi-fluorescence. The dyes andcorresponding resins are depicted in FIG. 59.

[0379] Fluorescence studies have been performed for each dye in solutionphase and attached to resin. FIG. 60 illustrates an example of theemission changes in 4 (part A.) and 7 (part B.) that result fromexposure to different solvent systems. The quantum yield of 4 diminishedin more polar protic media (i.e. ethanol); whereas, the quantum yield of4 increased in more hydrophobic environments (i.e. cyclohexane). Also,the Stoke's shift of each probe changed significantly between nonpolarand polar media. For example, the Stoke's shift of 4 (λ_(em)-λ_(abs)) in1:1 mixture of methanol and 1.0 M aqueous phosphate buffer was 221 nm,but the Stoke's shift of 4 was 80 nm in cyclohexane. 7 displayed similartrends, but the Stoke's shift from solvent to solvent was not asdramatic. The optical properties of 5-7 only varied slightly whencompared to their homogeneous analogs.

[0380] Of the three fluorophores, the solvatochromic properties ofcoumarin were not as dramatic when compared to dansyl and dapoxyl. 6 and7 displayed the largest Stoke's shifts. The emission wavelength for 5-7red shifted when placed in more polar solvents. However, when 6 wasplaced in water, the Stoke's shift was the same as in when placed incyclohexane as seen in FIG. 60. This trend was observed with eachfluoresently labeled resin, and may be explained by the fact that theseprobes are hydrophobic and that they may actually reside within thehydrophobic core of the PEG-PS resin when submerged in water.

[0381] In another example a selective chemosensor for ATP was found. Abead with a polyethylene-glycol base was attached via guanidinium to twolong polypeptide arms that were known to interact with the adenine groupof ATP, as depicted in FIG. 61. The tripeptide arms contained twoflourophore attachment sites for 5-carboxyfluorescein (fluorescein), andan attachment site for 7-diethylaminocoumarin-3-carboxylic acid(coumarin) located on the terminal end of the lysine that was attachedto the core structure. The fluorophores act as receptors for the desiredanalyte. The fluorophores also act as indicators to signal changes inthe environment before and after the addition of analytes.

[0382] Fluorescently labeled N-methylanthraniloyl-ATP were chosen toscreen for ATP receptors. Sequences of amino acids were linked astripeptides and equilibrated with a buffer. The resin was transferred toa microscope slide and illuminated with UV light. The results yielded 6sequences with active beads that displayed fluorescent activity, and 3sequences with inactive beads where there was no detectable fluorescentactivity.

[0383] Three of the 6 active beads, and 1 of the 3 inactive beads werearbitrarily chosen to react with ATP (Sequences below in bold). When thefluorescein and coumarin were excited there was no detectable differencein the FRET upon addition of ATP. This may be due to there being anaverage distance between the fluorophores within the beads which doesnot significantly change upon binding ATP. However, all but one activebead (Thr-Val-Asp) exhibited a fluorescence modulation upon excitationof fluorescein. The lack of response from an active bead shows thatscreening against a derivatized analyte (MANT-ATP in this case) will notguarantee that the active beads are successful sensors when synthesizedwith attached fluorophores. Either this active bead binds the MANTprotion of MANT-ATP or there is no significant microenvironment changearound the fluorophores of the Thr-Val-Asp receptor upon binding ATP.Active Beads Inactive Beads His-Ala-Asp His-Phe-Gly Glu-Pro-ThrSer-Ala-Asp Thr-Val-Asp Trp-Asn-Glu Met-Thr-His Asp-Ala-Asp Ser-Tyr-Ser

[0384] A large spectral response upon addition of ATP was observed withthe Ser-Tyr-Ser sequence in the active bead. The increase in fluoresceinemission is possibly due to a higher local pH around the fluoresceinupon binding of ATP. Further studies were performed with the Ser-Tyr-Sersequence and analytes, AMP, and GTP, which are structurally similar toATP. This peptidic library member exhibited very high detectionselectivity for ATP over these structurally similar potentiallycompeting analytes. The lack of response to AMP suggests the necessityfor triphosphates to bind strongly to the guanidinium entities of thereceptor, while the lack of response to GTP indicates the specificityfor nucleotide bases imparted by the tripeptide arms. The combination ofserine and tyrosine suggests -stacking between the phenol of tyr andadenine and hydrogen bonding interactions between the serine OH and/orthe ribose or adenine. These studies have demonstrated that the union ofa proven core with combinatorial methods, followed by the attachment offluorophores, can create resin bound chemosensors with excellentselectivity.

[0385] As described above, a particle, in some embodiments, possessesboth the ability to interact with the analyte of interest and to createa modulated signal. In one embodiment, the particle may include receptormolecules which undergo a chemical change in the presence of the analyteof interest. This chemical change may cause a modulation in the signalproduced by the particle. Chemical changes may include chemicalreactions between the analyte and the receptor. Receptors may includebiopolymers or organic molecules. Such chemical reactions may include,but are not limited to, cleavage reactions, oxidations, reductions,addition reactions, substitution reactions, elimination reactions, andradical reactions.

[0386] In one embodiment, the mode of action of the analyte on specificbiopolymers may be taken advantage of to produce an analyte detectionsystem. As used herein biopolymers refers to natural and unnatural:peptides, proteins, polynucleotides, and oligosaccharides. In someinstances, analytes, such as toxins and enzymes, will react withbiopolymer such that cleavage of the biopolymer occurs. In oneembodiment, this cleavage of the biopolymer may be used to produce adetectable signal. A particle may include a biopolymer and an indicatorcoupled to the biopolymer. In the presence of the analyte the biopolymermay be cleaved such that the portion of the biopolymer which includesthe indicator may be cleaved from the particle. The signal produced fromthe indicator is then displaced from the particle. The signal of thebead will therefore change thus indicating the presence of a specificanalyte.

[0387] Proteases represent a number of families of proteolytic enzymesthat catalytically hydrolyze peptide bonds. Principal groups ofproteases include metalloproteases, serine porteases, cysteine proteasesand aspartic proteases. Proteases, in particular serine proteases, areinvolved in a number of physiological processes such as bloodcoagulation, fertilization, inflammation, hormone production, the immuneresponse and fibrinolysis.

[0388] Numerous disease states are caused by and may be characterized byalterations in the activity of specific proteases and their inhibitors.For example emphysema, arthritis, thrombosis, cancer metastasis and someforms of hemophilia result from the lack of regulation of serineprotease activities. In case of viral infection, the presence of viralproteases have been identified in infected cells. Such viral proteasesinclude, for example, HIV protease associated with AIDS and NS3 proteaseassociated with Hepatitis C. Proteases have also been implicated incancer metastasis. For example, the increased presence of the proteaseurokinase has been correlated with an increased ability to metastasizein many cancers.

[0389] In one embodiment, the presence of a protease may be detected bythe use of a biopolymer coupled to a polymeric resin. For the detectionof proteases, the biopolymer may be a protein or peptide. Methods forsynthesizing and/or attaching a protein or peptides to a polymeric resinare described, for example, in U.S. Pat. No. 5,235,028 which isincorporated herein by reference. “Proteins” and “peptides” are hereindefined as chains of amino acids whose α-carbons are linked throughpeptide bonds formed by a condensation reaction between the a carboxylgroup of one amino acid and the amino group of another amino acid.Peptides also include peptide mimetics such as amino acids joined by anether as opposed to an amide bond.

[0390] The term “protease binding site” as used herein refers to anamino acid sequence that may be recognized and cleaved by a protease.The protease binding site contains a peptide bond that is hydrolyzed bythe protease and the amino acid residues joined by this peptide bond aresaid to form the cleavage site. The protease binding site andconformation determining regions form a contiguous amino acid sequence.The protease binding site may be an amino acid sequence that isrecognized and cleaved by a particular protease. It is well known thatvarious proteases may cleave peptide bonds adjacent to particular aminoacids. Thus, for example, trypsin cleaves peptide bonds following basicamino acids such as arginine and lysine and chymotrypsin cleaves apeptide bonds following large hydrophobic amino acid residues such astryptophan, phenylalanine, tyrosine and leucine. The serine proteaseelastase cleaves peptide bonds following small hydrophobic residues suchas alanine. A particular protease, however, may not cleave every bond ina protein that has the correct adjacent amino acid. Rather, theproteases may be specific to particular amino acid sequences which serveas protease binding sites for each particular protease. Any amino acidsequence that comprises a protease binding site and may be recognizedand cleaved by a protease is a suitable protease receptor. Knownprotease binding sites and peptide inhibitors of proteases posses aminoacid sequences that are recognized by the specific protease they arecleaved by or that they inhibit. Thus known substrate and inhibitorsequences provide the basic sequences suitable for use as a proteasereceptor. A number of protease substrates and inhibitor sequencessuitable for use as protease binding sites are described in U.S. Pat.No. 6,037,137 which is incorporated herein by reference. One of skillwill appreciate that the protease substrates listed in U.S. Pat. No.6,037,137 is not a complete list and that other protease substrates orinhibitor sequences may be used.

[0391] Proteases (e.g., botulinum and tetanus toxins) cleave peptidebonds at specific sequence sites on the proteins that “dock”neurotransmitter secretory vesicles to their cellular release sites(FIGS. 45A, 45B). When one or more of these proteins is degraded in thisfashion, secretion is blocked and paralysis results (FIG. 45C). It isknown that relatively low molecular weight peptides (˜15-35 amino acids)based on the normal protein substrates of the botulinum toxins can berapidly cleaved in solution by a toxin in a manner similar to thefull-length protein. Such experiments have been described by Schmidt, J.J.; Stafford, R. G.; Bostian, K. A. “Type A botulinum neurotoxinproteolytic activity: development of competitive inhibitors andimplications for substrate specificity at the S₁′ binding subsite” FEBSLett., 1998, 435, 61-64 and Shone, C. C.; Roberts, A. K. “Peptidesubstrate specificity and properties of the zinc-endopeptidase activityof botulinum type B neurotoxin” Eur. J. Biochem., 1994, 225, 263-270,both of which are incorporated herein by reference as if set forthherein. It has also been demonstrated that these peptide substrates canretain high levels of activity for both botulinum and tetanus toxinseven when chemically modified by amino acid substitutions andfluorescence labeling (See also Soleihac, J. -M.; Comille, F.; Martin,L.; Lenoir, C.; Foumie-Zaluski, M. -C.; Roques, B. P. “A sensitive andrapid fluorescence-based assay for determination of tetanus toxinpeptidase activity” Anal. Biochem., 1996, 241, 120-127 and Adler, M.;Nicholson, J. D.; Hackley, B. E., Jr. “Efficacy of a novelmetalloprotease inhibitor on botulinum neurotoxin B activity” FEBSLett., 1998, 429, 234-238 both of which are incorporated herein byreference).

[0392] For newly discovered proteases, or proteases of which theprotease recognition sequence is not known, a suitable amino acidsequence for use as the protease binding site may be determinedexperimentally. The synthesis of libraries of peptides and the use ofthese libraries to determine a protease binding sequence for aparticular protease is described in U.S. Pat. No. 5,834,318 which isincorporated herein by reference. Generally, combinatorial librariescomposed of between about 2 to about 20 amino acids may be synthesized.These libraries may be used to screen for an interaction with theprotease. Analysis of the sequences that bind to the protease may beused to determine potential binding sequences for use as a receptor forthe protease.

[0393] The interaction of the receptor with a protease may be indicatedby an indicator molecule coupled to the receptor or the polymeric resin.In one embodiment, the indicator may be a chromophore or a fluorophore.A fluorophore is a molecule that absorbs light at a characteristicwavelength and then re-emits the light most typically at acharacteristic different wavelength. Fluorophores include, but are notlimited to rhodamine and rhodamine derivatives, fluorescein andfluorescein derivatives, coumarins and chelators with the lanthanide ionseries. A chromophore is a molecule which absorbs light at acharacteristic wavelength, but does not re-emit light.

[0394] In one embodiment, a peptide containing the cleavage sequence isimmobilized through a covalent or strong non-covalent bond to anaddressable site on a sensor array. In one embodiment, this may beaccomplished by coupling the peptide to a polymeric resin, as describedabove. The polymeric resin may be positioned in a cavity of a sensorarray, such as the sensor arrays described above. In some embodiments,different peptides containing different cleavage sequences for thevarious proteases may be immobilized at different array positions. Asample containing one or more proteases may be applied to the array, andpeptide cleavage may occur at specific array addresses, depending on thepresence of particular proteases. Alternatively, different peptidescontaining different cleavage sequences may be coupled to a singlepolymeric bead. In this manner, a single bead may be used to analyzemultiple proteases.

[0395] A variety of signaling mechanisms for the above describedcleavage reactions may be used. In an embodiment, a fluorescent dye anda fluorescence quencher may be coupled to the biopolymer on oppositesides of the cleavage site. The fluorescent dye and the fluorescencequencher may be positioned within the Förster energy transfer radius.The Förster energy transfer radius is defined as the maximum distancebetween two molecules in which at least a portion of the fluorescenceenergy emitted from one of the molecules is quenched by the othermolecule. Förster energy transfer has been described above. Beforecleavage, little or no fluorescence may be generated by virtue of themolecular quencher. After cleavage, the dye and quencher are no longermaintained in proximity of one another, and fluorescence may be detected(FIG. 62A). The use of fluorescence quenching is described in U.S. Pat.No. 6,037,137 which is incorporated herein by reference. Furtherexamples of this energy transfer are described in the following papers,all of which are incorporated herein by reference: James, T. D.;Samandumara, K. R. A.; Iguchi, R.; Shinkai, S. J. Am. Chem. Soc. 1995,117, 8982. Murukami, H.; Nagasaki, T.; Hamachi, I.; Shinkai, S.Tetrahedron Lett., 34, 6273. Shinkai, S.; Tsukagohsi, K.; Ishikawa, Y.;Kunitake, T. J. Chem. Soc. Chem. Commun. 1991, 1039. Kondo, K.; Shiomi,Y.; Saisho, M.; Harada, T.; Shinkai, S. Tetrahedron. 1992, 48, 8239.Shiomi, Y.; Kondo, K.; Saisho, M.; Harada, T.; Tsukagoshi, K.; Shinkai,S. Supramol. Chem. 1993, 2, 11. Shiomi, Y.; Saisho, M.; Tsukagoshi, K.;Shinkai, S. J. Chem. Soc. Perkin Trans I 1993, 2111. Deng, G.; James, T.D.; Shinkai, S. J. Am. Chem. Soc. 1994, 116, 4567. James, T. D.; Harada,T.; Shinkai, S. J. Chem. Soc. Chem. Commun. 1993, 857. James, T. D.;Murata, K.; Harada, T.; Ueda, K.; Shinkai, S. Chem. Lett. 1994, 273.Ludwig, R.; Harada, T.; Ueda, K.; James, T. D.; Shinkai, S. J. Chem.Soc. Perkin Trans 2. 1994, 4, 497. Sandanayake, K. R. A. S.; Shinkai, S.J. Chem. Soc., Chem. Commun. 1994, 1083. Nagasaki, T.; Shinmori, H.;Shinkai, S. Tetrahedron Lett. 1994, 2201. Murakami, H.; Nagasaki, T.;Hamachi, I.; Shinkai, S. J. Chem. Soc. Perkin Trans 2. 1994, 975.Nakashima, K.; Shinkai, S. Chem. Lett. 1994, 1267. Sandanayake, K. R. A.S.; Nakashima, K.; Shinkai, S. J. Chem. Soc. 1994, 1621. James, T. D.;Sandanayake, K. R. A. S.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1994,477. James, T. D.; Sandanayake, K. R. A. S.; Angew. Chem., Int. Ed. Eng.1994, 33, 2207. James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S.Nature, 1995, 374, 345.

[0396] The fluorophores may be linked to the peptide receptor by any ofa number of means well known to those of skill in the art. In anembodiment, the fluorophore may be linked directly from a reactive siteon the fluorophore to a reactive group on the peptide such as a tenninalamino or carboxyl group, or to a reactive group on an amino acid sidechain such as a sulfur, an amino, a hydroxyl, or a carboxyl moiety. Manyfluorophores normally contain suitable reactive sites. Alternatively,the fluorophores may be derivatized to provide reactive sites forlinkage to another molecule. Fluorophores derivatized with functionalgroups for coupling to a second molecule are commercially available froma variety of manufacturers. The derivatization may be by a simplesubstitution of a group on the fluorophore itself, or may be byconjugation to a linker. Various linkers are well known to those ofskill in the art and are discussed below.

[0397] The fluorogenic protease indicators may be linked to a solidsupport directly through the fluorophores or through the peptidebackbone comprising the indicator. In embodiments where the indicator islinked to the solid support through the peptide backbone, the peptidebackbone may comprise an additional peptide spacer. The spacer may bepresent at either the amino or carboxyl terminus of the peptide backboneand may vary from about 1 to about 50 amino acids, preferably from 1 toabout 20 and more preferably from 1 to about 10 amino acids in length.The amino acid composition of the peptide spacer is not critical as thespacer just serves to separate the active components of the moleculefrom the substrate thereby preventing undesired interactions. However,the amino acid composition of the spacer may be selected to provideamino acids (e.g. a cysteine or a lysine) having side chains to which alinker or the solid support itself, is easily coupled. Alternatively thelinker or the solid support itself may be attached to the amino terminusof or the carboxyl terminus.

[0398] In an embodiment, the peptide spacer may be joined to the solidsupport by a linker. The term “linker”, as used herein, refers to amolecule that may be used to link a peptide to another molecule, (e.g. asolid support, fluorophore, etc.). A linker is a hetero orhomobifunctional molecule that provides a first reactive site capable offorming a covalent linkage with the peptide and a second reactive sitecapable of forming a covalent linkage with a reactive group on the solidsupport. Linkers as use din these embodiments are the same as thepreviously described linkers.

[0399] In an embodiment, a first fluorescent dye and a secondfluorescent dye may be coupled to the biopolymer on opposite sides ofthe cleavage site. Before cleavage, a FRET (fluorescence resonanceenergy transfer) signal may be observed as a long wavelength emission.After cleavage, the change in the relative positions of the two dyes maycause a loss of the FRET signal and an increase in fluorescence from theshorter-wavelength dye (FIG. 62B). Examples of solution phase FRET havebeen described in Förster, Th. “Transfer Mechanisms of ElectronicExcitation:, Discuss. Faraday Soc., 1959, 27, 7; Khanna, P. L., Ullman,E. F. “4′,5′-Dimethoxyl-6-carboxyfluorescein: A novel dipole-dipolecoupled fluorescence energy transfer acceptor useful for fluorescenceimmunoassays”, Anal. Biochem. 1980, 108, 156; and Morrison, L. E. “Timeresolved Detection of Energy Transfer: Theory and Application toImmunoassays”, Anal. Biochem. 1998, 174, 101, all of which areincorporated herein by reference.

[0400] In another embodiment, a single fluorescent dye may be coupled tothe peptide on the opposite side of the cleavage site to the polymericresin. Before cleavage, the dye is fluorescent, but is spatiallyconfined to the attachment site. After cleavage, the peptide fragmentcontaining the dye may diffuse from the attachment site (e.g., topositions elsewhere in the cavity) where it may be measured with aspatially sensitive detection approach, such as confocal microscopy(FIG. 62C). Alternatively, the solution in the cavities may be flushedfrom the system. A reduction in the fluorescence of the particle wouldindicate the presence of the analyte (e.g., a protease).

[0401] In another embodiment, a single indicator (e.g., a chromophore ora fluorophore) may be coupled to the peptide receptor on the side of thecleavage site that remains on the polymeric resin or to the polymericresin at a location proximate to the receptor. Before cleavage theindicator may produce a signal that reflects the microevironmentdetermined by the interaction of the receptor with the indicator.Hydrogen bonding or ionic substituents on the indicator involved inanalyte binding have the capacity to change the electron density and/orrigidity of the indicator, thereby changing observable spectroscopicproperties such as fluorescence quantum yield, maximum excitationwavelength, or maximum emission wavelength for fluorophores orabsorption spectra for chromophores. When the peptide receptor iscleaved, the local pH and dielectric constants of the beads change, andthe indicator may respond in a predictable fashion. An advantage to thisapproach is that it does not require the dissociation of a preloadedfluorescent ligand (limited in response time by k_(off)). Furthermore,several different indicators may be used with the same receptor.Different beads may have the same receptors but different indicators,allowing for multiple testing for the presence of proteases.Alternatively, a single polymeric resin may include multiple dyes alongwith a single receptor. The interaction of each of these dyes with thereceptor may be monitored to determine the presence of the analyte.

[0402] Nucleases represent a number of families of enzymes thatcatalytically hydrolyze the phosphodiester bonds of nucleic acids.Nucleases may be classified according to the nucleic acid that they arespecific for. Ribonucleases (“RNases”) are specific for ribonucleicacids while deoxyribonucleases (“DNases”) are specific fordeoxyribonucleic acids. Some enzymes will hydrolyze both ribonucleicacids and deoxyribonucleic acids. Nucleases may also be classifiedaccording to their point of attack upon the nucleic acid. Nucleases thatattack the polymer at either the 3′ terminus or the 5′ terminus areknown as exonucleases. Nucleases that attack the nucleic acid within thechain are called endonucleases.

[0403] Restriction enzymes recognize short polynucleotide sequences andcleave double-stranded nucleic acids at specific sites within oradjacent to these sequences. Approximately 3,000 restriction enzymes,recognizing over 230 different nucleic acid sequences, are known. Theyhave been found mostly in bacteria, but have also been isolated fromviruses, archaea and eukaryotes. Because many of these restrictionenzymes are only found in a particular organism, nucleic acids may beused as a receptor to determine if a particular organism is present in asample by analyzing for restriction enzymes. Restriction endonucleasesspecifically bind to nucleic acids only at a specific recognitionsequence that varies among restriction endonucleases. Since restrictionenzymes only cut nucleic acids in the vicinity of the recognitionsequence, a receptor may be designed that includes the recognitionsequence for the nuclease being investigated.

[0404] Most nucleases bind to and act on double strandeddeoxyribonucleic acid (“DNA”). Restriction endonucleases are typicallysymmetrical dimers. Each monomeric unit binds to one strand of DNA andrecognizes the first half the DNA recognition sequence. Each monomeralso typically cuts one strand of DNA. Together, the dimer recognizes apalindromic DNA sequence and cuts both strands of DNA symmetricallyabout the central point in the palindromic sequence. Typically, eachmonomer of the restriction endonucleases needs at least two specificnucleotides that it recognizes, though in a few cases a restrictionendonuclease monomer only needs to bind to one specific nucleotide andtwo others with less specificity. This means that restrictionendonucleases may recognize a sequence of 4 nucleotides at a minimum,and generally recognize sequences that contain an even number ofnucleotides (since the same sites are recognized by each monomer.Restriction endonucleases are known that recognize 4, 6, or 8nucleotides, with only a few 8-cutters known. Some restrictionendonucleases bind to recognition sequences that have an odd number ofnucleotides (typically this is 5 or 7) with the central nucleotidespecifically recognized or with some or strict specificity for a centralbase pair. The origin and sequence specificity of hundreds ofrestriction endonucleases are known and can be found from catalogsavailable from New England Biolabs, Boston, Mass.; Life Technologies,Rockville, Md.; Promega Scientific, Madison, Wis., Rouche MolecularBiochemicals, Indianapolis, Ind.

[0405] In one embodiment, the presence of a nuclease may be detected bythe use of a polynucleotide coupled to a polymeric resin. For thedetection of nucleases, the polynucleotide may be a double strandeddeoxyribonucleic acid or a ribonucleic acid. Methods for synthesizingand/or attaching a polynucleotide to a polymeric resin are described,for example, in U.S. Pat. No. 5,843,655 which is incorporated herein byreference. “Polynucleotides” are herein defined as chains ofnucleotides. The nucleotides are linked to each other by phosphodiesterbonds. “Deoxyribonucleic acid” is composed of deoxyribonucleotideresidues, while “Ribonucleic acid” is composed of ribonucleotideresidues.

[0406] The term “nuclease binding site” as used herein refers to apolynucleotide sequence that may be recognized and cleaved by anuclease. The nuclease binding site contains a phosphodiester bond thatis cleaved by the nuclease and the polynucleotide residues joined bythis phosphodiester bond are said to form the cleavage site.

[0407] For newly discovered nucleases, or nucleases of which thenuclease recognition sequence is not known, a suitable polynucleotidesequence for use as the nuclease binding site may be determinedexperimentally. Generally, combinatorial libraries of polynucleotidescomposed of between about 2 to about 20 nucleotides may be synthesized.The synthesis of such libraries is described, for example, in U.S. Pat.No. 5,843,655 which is incorporated herein by reference. These librariesmay be used to screen for an interaction with the nuclease. Analysis ofthe sequences that bind to the nuclease may be used to determinepotential binding sequences for use as a receptor for the nuclease.

[0408] The interaction of the receptor with a nuclease may be indicatedby an indicator molecule coupled to the receptor or the polymeric resin.In one embodiment, the indicator may be a chromophore or a fluorophore.

[0409] In one embodiment, a polynucleotide containing the nucleasebinding sequence is immobilized through a covalent or strongnon-covalent bond to an addressable site on a sensor array. In oneembodiment, this may be accomplished by coupling or synthesizing thepolynucleotide on a polymeric resin, as described above. The polymericresin may be positioned in a cavity of a sensor array, such as thesensor arrays described above. In some embodiments, differentpolynucleotides containing different cleavage sequences for the variousnucleases may be immobilized at different array positions. A samplecontaining one or more nucleases may be applied to the array, andpolynucleotide cleavage may occur at specific array addresses, dependingon the presence of particular nucleases. Alternatively, differentpolynucleotides containing different cleavage sequences may be coupledto a single polymeric bead. In this manner, a single bead may be used toanalyze multiple nucleases.

[0410] A variety of signaling mechanisms for the above describedcleavage reactions may be used. In an embodiment, a fluorescent dye anda fluorescence quencher may be coupled to the polynucleotide on oppositesides of the cleavage site. The fluorescent dye and the fluorescencequencher may be positioned within the Förster energy transfer radius.Before cleavage, little or no fluorescence may be generated by virtue ofthe molecular quencher. After cleavage, the dye and quencher are nolonger maintained in proximity of one another, and fluorescence may bedetected (FIG. 62A).

[0411] The fluorophores may be linked to the polynucleotide receptor byany of a number of means well known to those of skill in the art.Examples of methods of attaching fluorophores and dyes topolynucleotides are described in U.S. Pat. Nos. 4,855,225; 5,188,934,and 5,366,860 all of which are incorporated herein by reference.

[0412] In another embodiment, a first fluorescent dye and a secondfluorescent dye may be coupled to the polynucleotide receptor onopposite sides of the cleavage site. Before cleavage, a FRET(fluorescence resonance energy transfer) signal may be observed as along wavelength emission. After cleavage, the change in the relativepositions of the two dyes may cause a loss of the FRET signal and anincrease in fluorescence from the shorter-wavelength dye (FIG. 62B).

[0413] In another embodiment, a single fluorescent dye may be coupled tothe polynucleotide receptor on the opposite side of the cleavage site tothe polymeric resin. Before cleavage, the dye is fluorescent, but isspatially confined to the attachment site. After cleavage, the nucleicacid fragment containing the dye may diffuse from the attachment site(e.g., to positions elsewhere in the cavity) where it may be measuredwith a spatially sensitive detection approach, such as confocalmicroscopy (FIG. 62C). Alternatively, the solution in the cavities maybe flushed from the system. A reduction in the fluorescence of theparticle would indicate the presence of the analyte (e.g., a nuclease).

[0414] In another embodiment, depicted in FIG. 62D, a single indicator(e.g., a chromophore or a fluorophore) may be coupled to thepolynucleotide receptor on the side of the cleavage site that remains onthe polymeric resin or to the polymeric resin at a location proximate tothe polynucleotide receptor. Before cleavage the indicator may produce asignal that reflects the microevironment determined by the interactionof the receptor with the indicator. Hydrogen bonding or ionicsubstituents on the indicator involved in analyte binding have thecapacity to change the electron density and/or rigidity of theindicator, thereby changing observable spectroscopic properties such asfluorescence quantum yield, maximum excitation wavelength, or maximumemission wavelength for fluorophores or absorption spectra forchromophores. When the polynucleotide receptor is cleaved, the local pHand dielectric constants of the beads change, and the indicator mayrespond in a predictable fashion. An advantage to this approach is thatit does not require the dissociation of a preloaded fluorescent ligand(limited in response time by k_(off)). Furthermore, several differentindicators may be used with the same receptor. Different beads may havethe same receptors but different indicators, allowing for multipletesting for the presence of nucleases. Alternatively, a single polymericresin may include multiple dyes along with a single receptor. Theinteraction of each of these dyes with the receptor may be monitored todetermine the presence of the analyte.

[0415] In another embodiment, polynucleotide receptors may be used todetermine the presence of other types of analytes. It some instances,polynucleotide receptors will bind to small organic molecules. Thesesmall organic molecules may disrupt the action of nucleases upon thepolynucleotide receptor. Typically, the small molecules will occupy thepreferred binding site of the nuclease, inhibiting the action of thenuclease on the polynucleotide. Thus the presence of a small organicmolecule, which is known to bind to a specific polynucleotide, may bedetected by the observation of reduced nuclease activity at the specificpolynucleotide. An analogous methodology may be applied to apeptide-protease reaction.

[0416] In another embodiment, oligosaccharides may also be used todetermine the presence of analytes. In a system similar to thosedescribed above for peptides and polynucleotides, oligosaccharides maybe coupled to a polymeric resin. In the presence of oligosaccharidecleaving agents (e.g., enzymes such as amylase, an enzyme that cleaves along saccharide polymer and disaccharide cleaving enzymes such asinvertase, β-galactosidase, and lactase, to name a few) theoligosaccharide may be cleaved. The cleavage of the oligosaccharide maybe used to generate a signal. Methods for synthesizing and/or attachingoligosaccharides to a polymeric resin are described, for example, inU.S. Pat. Nos. 5,278,303 and 5,616,698 which are incorporated herein byreference.

[0417] In another embodiment, an analyte may cause a change to abiopolymer, but not necessarily cleavage of the biopolymer, when theanalyte interacts with the biopolymer. The induced change may cause adetectable signal to be generated. Typically, the binding or associationability of an indicator molecule with a biopolymer is dependent upon thestructure of the biopolymer. If the structure of the biopolymer isaltered, the association of an indicator molecule may be significantlyaltered. Such a change may be accompanied by a change in the signalproduced by the indicator. For biopolymers many different types ofenzymes may induce a variety of structural changes to the biopolymerwhich may alter the binding site of an associated indicator molecule.Such changes may occur without cleavage of the biopolymer.

[0418] Alternatively, an indicator and a biopolymer may be coupled to apolymeric bead. The biopolymer may undergo a chemical reaction in thepresence of an analyte. This chemical reaction may also induce a changein the chemical structure of the indicator. The change in the chemicalstructure of the indicator may lead to a detectable change in theoptical properties of the particle, signaling the presence of theanalyte.

[0419] In one example, NAD and glucose may be coupled to a polymericbead. This system may be used to detect the presence of an carbohydratemodifying enzyme. For example, the system may be used to detect thepresence of glucose dehydrogenase. In the presence of glucosedehydrogenase, glucose may be consumed, and in the process would convertthe coupled NAD into NADH. NADH has both different UV absorbance anddifferent fluorescence properties from NAD. These differences may beused to signal the presence of glucose dehydrogenase in a fluid sample.Many other types of enzymes may be detected in a similar manner..

[0420] In an example, the protease trypsin was analyzed using animmobilized “sacrificial receptor” that is cleaved by trypsin, an eventthat results in modulation of a fluorescence signal. In an embodiment ofa protease assay, a peptide that may be cleaved between two amino acidsby the enzyme trypsin was immobilized. This immobilization wasaccomplished by first conjugating many streptavidin molecules toaldehyde-activated 6% agarose beads using a reductive aminationprocedure. A biotin chemical group attached to the amino-terminus of thepeptide was strongly bound by the immobilized streptavidin molecules,thereby immobilizing the peptide chains. A fluorescein group wasattached to the carboxyl-terminus of the peptide, thereby making thebead highly fluorescent. Importantly, the immobilized peptide contains acleavage site recognized by trypsin between the biotin attachment siteand the fluorescein, so that exposure of the bead to trypsin analytecauses release of fluorescent peptide fragments from the bead. Thisrelease may be visualized either as a decrease in the fluorescence atthe bead, or by an increase in the fluorescence of the surroundingsolution (see FIG. 63).

[0421] Transmitting Chemical Information Over a Computer Network

[0422] Herein we describe a system and method for the collection andtransmission of chemical information over a computer network. Thesystem, in some embodiments, includes an analyte detection device(“ADD”) operable to detect one or more analytes or mixtures of analytesin a fluid containing one or more analytes, and computer hardware andsoftware operable to send and receive data over a computer network toand from a client computer system.

[0423] Chemical information refers to any data representing thedetection of a specific chemical or a combination of chemicals. Thesedata may include, but are not limited to chemical identification,chemical proportions, or various other forms of information related tochemical detection. The information may be in the form of raw data,including binary or alphanumeric, formatted data, or reports. In someembodiments, chemical information relates to data collected from ananalyte detection device. Such data includes data related to the colorof the particles included on the analyte detection device. The chemicalinformation collected from the analyte detection device may include rawdata (e.g., a color, RBG data, intensity at a specific wavelength) etc.Alternatively the data may be analyzed by the analyte detection deviceto determine the analytes present. The chemical information may includethe identities of the analytes detected in the fluid sample. Theinformation may be encrypted for security purposes.

[0424] In one embodiment, the chemical information may be in LogicalObservation Identifiers Names and Codes (LOINC) format. The LOINC formatprovides a standard set of universal names and codes for identifyingindividual laboratory results (e.g. hemoglobin, serum sodiumconcentration), clinical observations (e.g. discharge diagnosis,diastolic blood pressure) and diagnostic study observations, (e.g.PR-interval, cardiac echo left ventricular diameter, chest x-rayimpression).

[0425] More specifically, chemical information may take the form of datacollected by the analyte detection system. As described above, ananalyte detection system may include a sensor array that includes aparticle or particles. These particles may be configured to produce adetectable signal in response to the presence or absence of an analyte.The signal may be detected using a detector. The detector may detect thesignal. The detector may also produce an output signal that containsinformation relating to the detected signal. The output signal may, insome embodiments be the chemical information.

[0426] In some embodiments, the detector may be a light detector and thesignal produced by the particles may be modulated light. The detectormay produce an output signal that is representative of the detectedlight modulation. The output signal may be representative of thewavelength of the light signal detected. Alternatively, the outputsignal may be representative of the strength of the light signaldetected. In other embodiments, the output signal may include bothwavelength and strength of signal information.

[0427] In some embodiments, use of a light source may not be necessary.The particles may rely on the use of chemiluminescence,thermoluminescence or piezoluminescence to provide a signal. In thepresence of an analyte of interest, the particle may be activated suchthat the particles At produce light. In the absence of an analyte, theparticles may not exhibit produce minimal or no light. The chemicalinformation may, therefore, be related to the detection or absence of alight produced by the particles, rather than modulated by the particles.

[0428] The detector output signal information may be analyzed byanalysis software. The analysis software may be configured to convertthe raw output data to chemical information that is representative ofthe analytes in the analyzed fluid system. The chemical information maybe either the raw data before analysis by the computer software or theinformation generated by processing of the raw data.

[0429] The term “computer system” as used herein generally describes thehardware and software components that in combination allow the executionof computer programs. The computer programs may be implemented insoftware, hardware, or a combination of software and hardware. Computersystem hardware generally includes a processor, memory media, andinput/output (I/O) devices. As used herein, the term “processor”generally describes the logic circuitry that responds to and processesthe basic instructions that operate a computer system. The term “memorymedium” includes an installation medium, e.g., a CD-ROM, floppy disks; avolatile computer system memory such as DRAM, SRAM, EDO RAM, Rambus RAM,etc.; or a non-volatile memory such as optical storage or a magneticmedium, e.g., a hard drive. The term “memory” is used synonymously with“memory medium” herein. The memory medium may comprise other types ofmemory or combinations thereof. In addition, the memory medium may belocated in a first computer in which the programs are executed, or maybe located in a second computer that connects to the first computer overa network. In the latter instance, the second computer provides theprogram instructions to the first computer for execution. In addition,the computer system may take various forms, including a personalcomputer system, mainframe computer system, workstation, networkappliance, Internet appliance, personal digital assistant (PDA),television system or other device. In general, the term “computersystem” can be broadly defined to encompass any device having aprocessor that executes instructions from a memory medium.

[0430] The memory medium preferably stores a software program orprograms for the reception, storage, analysis, and transmittal ofinformation produced by an Analyte Detection Device (ADD). The softwareprogram(s) may be implemented in any of various ways, includingprocedure-based techniques, component-based techniques, and/orobject-oriented techniques, among others. For example, the softwareprogram may be implemented using ActiveX controls, C++ objects,JavaBeans, Microsoft Foundation Classes (MFC), or other technologies ormethodologies, as desired. A central processing unit (CPU), such as thehost CPU, for executing code and data from the memory medium includes ameans for creating and executing the software program or programsaccording to the methods, flowcharts, and/or block diagrams describedbelow.

[0431] A computer system's software generally includes at least oneoperating system such as Windows NT, Windows 95, Windows 98, or WindowsME (all available from Microsoft Corporation); or Mac OS and Mac OS XServer (Apple Computer, Inc.), MacNFS (Thursby Software), PC MACLAN(Miramar Systems), or real time operating systems such as VXWorks (WindRiver Systems, Inc.), QNX (QNX Software Systems, Ltd.), etc. Theforegoing are all examples of specialized software programs that manageand provide services to other software programs on the computer system.Software may also include one or more programs to perform various taskson the computer system and various forms of data to be used by theoperating system or other programs on the computer system. Software mayalso be operable to perform the functions of an operating system (OS).The data may include but is not limited to databases, text files, andgraphics files. A computer system's software generally is stored innon-volatile memory or on an installation medium. A program may becopied into a volatile memory when running on the computer system. Datamay be read into volatile memory as the data is required by a program.

[0432] A server program may be defined as a computer program that, whenexecuted, provides services to other computer programs executing in thesame or other computer systems. The computer system on which a serverprogram is executing may be referred to as a server, though it maycontain a number of server and client programs. In the client/servermodel, a server program awaits and fulfills requests from clientprograms in the same or other computer systems. Examples of computerprograms that may serve as servers include: Windows NT (MicrosoftCorporation), Mac OS X Server (Apple Computer, Inc.), MacNFS (ThursbySoftware), PC MACLAN (Miramar Systems), etc

[0433] A web server is a computer system which maintains a web sitebrowsable by any of various web browser software programs. As usedherein, the term ‘web browser’ refers to any software program operableto access web sites over a computer network.

[0434] An intranet is a network of networks that is contained within anenterprise. An intranet may include many interlinked local area networks(LANs) and may use data connections to connect LANs in a wide areanetwork (WAN). An intranet may also include connections to the Internet.An intranet may use TCP/EP, HTTP, and other Internet protocols.

[0435] An extranet, or virtual private network, is a private networkthat uses Internet protocols and public telecommunication systems tosecurely share part of a business' information or operations withsuppliers, vendors, partners, customers, or other businesses. Anextranet may be viewed as part of a company's intranet that is extendedto users outside the company. An extranet may require security andprivacy. Companies may use an extranet to exchange large volumes ofdata, share product catalogs exclusively with customers, collaboratewith other companies on joint development efforts, provide or accessservices provided by one company to a group of other companies, and toshare news of common interest exclusively with partner companies.

[0436] Connection mechanisms included in a network may include copperlines, optical fiber, radio transmission, satellite relays, or any otherdevice or mechanism operable to allow computer systems to communicate.

[0437] As used herein, ADD refers to any device or instrument operableto detect one or more specific analytes or mixtures of analytes in afluid sample, wherein the fluid sample may be liquid, gaseous, solid, asuspension of a solid in a gas, or a suspension of a liquid in a gas.More particularly, an ADD includes a sensor array, light and detector asis described herein.

[0438] As illustrated in FIG. 64, an ADD 102 is operable to analyze afluid sample and detect one or more analytes in the sample, producingoutput data specifying the results of the detection process. ADD 102 maybe operable to connect to a computer network 104, such as the Internet.As used herein, “computer network” may refer to any type of intranet orextranet network which connects computers and/or networks of computerstogether, thereby providing connectivity between various systems forcommunication there between, using various network communicationprotocols, such as TCP/IP, FTP, HTTP, RTTPS, etc. ADD 102 may executesoftware to communicate with other computer systems connected to network104.

[0439] A client computer 106 may also be connected to network 104. Theclient system 106 may be a computer system, network appliance, Internetappliance, personal digital assistant (PDA) or other system. Clientcomputer system 106 may execute software to communicate with ADD 102,thus facilitating transmission of chemical data from the ADD 102 toclient computer system 106 and vice versa.

[0440] In one embodiment, the ADD may execute software operable totransmit chemical data via any of various communication protocols overthe network to one or more recipient client computer systems and toreceive responses from the recipient client computers. These protocolsmay include, but are not limited to, TCP/IP, FTP, HTTP, and HTTPS. Asstated above, the chemical information may be encrypted for securitypurposes.

[0441] As FIG. 65 illustrates, in step 110 an ADD 102 may be used toanalyze a chemical sample and detect one or more particular analytes orcombinations of analytes, producing output data comprising the resultsof the detection process. As stated above, this information may be in avariety of forms and formats, including binary, alphanumeric, reports,etc. In one embodiment, the ADD is configured to detect optical signalsproduced by the reaction of the analyte with a sensor array ofparticles. The optical signals may be converted to output datarepresentative of the optical signal.

[0442] In step 112 the chemical information may be transmitted overnetwork 104 to one or more client computer systems 106 using any of avariety of network communication protocols as described herein.

[0443] In step 114 one or more client computer systems 106 may eachoptionally transmit a response back to ADD 102. The response mayinclude, but is not limited to, a request for additional information, aconfirmation of received data, or a transmittal of chemical informationback to the ADD.

[0444] Some embodiments of the ADD include a light source, a sensorarray, and a detector. The sensor array, in some embodiments, is formedof a supporting member which is configured to hold a variety ofchemically sensitive particles (herein referred to as “particles”) in anordered array. The particles are, in some embodiments, elements whichwill create a detectable signal in the presence of an analyte. Theparticles may produce optical (e.g., absorbance or reflectance) orfluorescence/phosphorescent signals upon exposure to an analyte.Examples of particles include, but are not limited to, functionalizedpolymeric beads. The particles may include a receptor molecule coupledto a polymeric bead. The receptors, in some embodiments, are chosen forinteracting with analytes. The interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles,while allowing the passage of the appropriate wavelengths of light. Thesupporting member may include a plurality of cavities. The cavities maybe formed such that at least one particle is substantially containedwithin the cavity. Upon contact of the beads with a fluid sample, adetectable optical signal may be generated by the receptor molecules'reactions with the one or more analytes in the sample.

[0445] In an alternate form of the invention, ADD 102 may be operable toupload chemical data directly to a local computer system 108, forexample, by a communications link such as a serial data connection,wireless data link, modem, floppy drive, etc., as depicted in FIG. 66.Local computer system 108 may be connected to the computer network 104,as may be client computer system 106. The local computer system 108 mayhave software executable to transmit chemical information to the clientcomputer system 106 and to receive response information back from theclient computer system 106, and client computer system 106 may havesoftware executable to receive chemical information and to transmit aresponse back to local computer system 108 or to one or more receivingcomputer systems 107.

[0446] As FIG. 67 illustrates, in step 210 an ADD 102 may be used toanalyze a chemical sample and detect one or more particular analytes orcombinations of analytes, producing output data comprising the resultsof the detection process.

[0447] In step 212 chemical information may be uploaded to a localcomputer system 108, such as by a communication link as described above.Local computer system 108 is connected to the network 104 and may use asoftware program executable to transmit the chemical information overnetwork 104.

[0448] As shown in step 214, the chemical information may be transmittedover network 104 to one or more client computer systems 106 using any ofa variety of network communication protocols, such protocols beingfamiliar to one skilled in the network communication art.

[0449] In step 216 client computer system 106 may optionally transmit aresponse back to local computer system 106 over network 104, or to oneor more receiving computer systems 107.

[0450] As shown in FIG. 68, ADD 102 may connect to a server 302, eitherdirectly, as with a communication link, or remotely, via computernetwork 104. The server 302 is operable to receive and store thechemical information, and to make the chemical information available toclient computer systems 106 also connected to network 104. The server302 may be any of a variety of servers. For example, server 302 may be aweb server, wherein the server is operable to maintain a web site,accessible by client computer systems 106 with browser software. Theuser of client computer system 106 may view and/or download the chemicalinformation from server 302 using the browser software. As anotherexample, the server may be an FTP server, in which case the user ofclient computer system 106 may be able to transfer the chemicalinformation from server 302 to client computer system 106 using an FTPsoftware program. As yet another example, server 302 may allow remotelogin to an account by client computer system 106, wherein the accounthas been established for use by the user of client computer system. Theuser of client computer system 106 may then view, edit, or transfer thechemical information as needed. Client computer system 106 may thenoptionally transmit a response back to server 302, which may then beaccessed by the ADD. Client computer system 106 may also transmit theresponse information to one or more additional client computer systems107. In all of these embodiments, security measures may be employed toprotect the identity of the users, as well as the privacy and integrityof the information. Such security measures may include secure login,encryption, private communication lines, and other security measures.

[0451] As FIG. 69 illustrates, in step 310 an ADD 102 may be used toanalyze a chemical sample and detect one or more particular analytes orcombinations of analytes, producing output data comprising the resultsof the detection process.

[0452] In step 312 the chemical information may be uploaded to a server302, either directly, as by communication link, or via the computernetwork 104. There, the chemical information may be stored.

[0453] As described above, server 302 is connected to network 104, as isthe client computer system 106. In step 314 client computer system 106may connect to server 302 over network 104.

[0454] As shown in step 316, chemical information may be transmitted byserver 302 over the network to client computer system 106 using any of avariety of network communication protocols, such as TCP/IP, FIP, HTTP,HTTPS, etc.

[0455] In step 318 client computer system 106 may optionally transmitresponse information back to server 302, which then may be accessed byADD 102 to retrieve the response information, or to one or moreadditional client computer systems 107.

[0456] In one embodiment, server 302 is a web server operable tomaintain a web site. When a client computer system accesses the web siteof web server 120, web server 120 provides various data and informationto the client browser on client system 106, possibly including agraphical user interface (GUI) that displays the information,descriptions of the information, and/or other information that might beuseful to the users of the system.

[0457] In some embodiments, the ADD may include an electroniccontroller, as described herein. The electronic controller may allow theADD to be operated by a client computer that is coupled to theelectronic controller. The client computer may include software thatprovides the user information regarding the operation of the ADD. Theclient computer may allow the user of the client computer to issuecommands that allow operation of the ADD from the electronic controller.The issued commands may be converted to control signals. The controlsignals may be received by the electronic controller. The electroniccontroller may operate components of the ADD in response to the receivedcontrol signals.

[0458] The client computer may be coupled directly to the ADD.Alternatively, the client computer may be coupled to the ADD via acomputer network. In this embodiment, an operator may be in a differentlocation than the location of the ADD. By sending control signals overthe computer network, the operator may remotely control the operation ofthe ADD. The ADD may also be configured to transmit the obtainedchemical information back to the client computer via the computernetwork.

[0459] In another embodiment, the client computer may be coupled to theADD via a server, as described before. The client computer may beconfigured to receive and/or transmit information to the ADD. In oneembodiment, the ADD may be configured to receive control signals fromthe client computer via the server. The operation of the ADD may,therefore, be controlled via a client computer through a server. Asdiscussed before the ADD may also transmit chemical information back tothe client computer via the server.

[0460] In one embodiment, the ADD may be used to detect and identify oneor more analytes in the blood serum of an animal or person in a remotelocation. The ADD may be configured with the appropriate detectioncomponents and software to detect the presence of any of a great numberof different analytes. The serum sample may be processed by the ADD andthe results, the chemical information, transmitted to a client computersystem residing at a diagnosis center (e.g. a veterinary hospital ormedical office). There a medical expert may receive the chemicalinformation and interpret it to diagnose the probable cause and/orsource of the detected analytes. The medical expert may use thisinformation to make a diagnosis of the patients medical condition. Basedon the diagnosed medical condition, the medical expert may alsoprescribe medication for the treatment of the medical condition. Thisinformation may be transmitted back to the ADD over a computer networkor a server. The information may also be transmitted to other clientcomputer systems that are linked using a computer network or a server.For example, the medical expert may transmit a prescription to the ADDand to a client computer system at a pharmacy, which may then fill theprescription.

[0461] In another example of the use of an embodiment of the invention,ADD 102 is used to detect pollutants in a water supply, e.g. a remotelake or stream. ADD 102 processes the water sample, and the resultingdetection information is transmitted to the web server 302 at anEnvironmental Monitoring station. The pollution information may includeboth identification and concentrations of the chemicals detected. There,the information may be input into a software program which updates a mapof area waterways with pollution information superimposed thereon. Theupdated map may be displayed on a web site for use by interestedparties.

[0462] In another embodiment, the invention may be used for home drugmetabolite tracking. Detection and measurement of blood and urinecomponents by ADD 102 may be used to track the appearance anddisappearance of various drug components after dosing. The user of ADD102 may upload the test results to a client computer system 106 used bya health care professional. The upload may be accomplished either bytransferring the information to a local computer system, thentransmitting over network 104 to client computer system 106 of thehealth professional, or directly from ADD 102 to client system 106 overnetwork 104 (e.g., through an internal modem similar to those used inPDAs or other hand-held computing devices). Results of the tests may beexamined either by human or software, and recommendations made to eithercontinue current drug protocol or to modify dosing to achieve a desiredmetabolite profile. These recommendations may then be transmitted backto the user of ADD 102, either via the local computer system, ordirectly, depending upon the ADD communication capabilities. Throughthis method, accurate determinations of doses needed to achieveeffective treatment while avoiding dangerous over-medication may bepossible. This offers a revolutionary change from current approaches, inwhich most or all people in a population are treated identically,regardless of ethnicity, gender, age, and medication with other drugs.Some studies indicate that effective and toxic dose levels can varysignificantly for these different subgroups of patients. By providing asimple and fast means for frequent metabolite analysis and evaluation,network uploading of ADD detection results, and possible subsequentdownloading of recommendations can open fundamentally new ways to treatpatients.

[0463] According to another embodiment, the method and system may beused for home blood component analysis by a patient. Similar to drugmetabolite tracking, the results of analyses for natural bloodcomponents (e.g., glucose, insulin, cholesterol (LDLs/HDLs),triglycerides, prostate-specific antigen, and other indicators of healthstate) may be uploaded to a client computer system 106 of a health careprofessional. Examination of test results could then be used fordiagnosis, or at least early-warning screening for possible pathologies.Recommendations for action (e.g., drug use or scheduling of appointment)may then be transmitted back to the patient's ADD 102 or local computersystem 106 or phoned to the patient. Again, the potential for simple,fast, and frequent measurements may provide safeguards for patients incertain risk groups (e.g., diabetes), who would otherwise need to makefrequent trips to the lab or at the minimum would otherwise have tomanually call in/email home test results—a far less reliable approachthan automated uploading of the chemical information by the ADDfollowing its analysis.

[0464] Another embodiment of the invention pertains to field-testing ofenvironmental conditions. Automated sensing of environmental conditions,including the presence of natural chemicals, industrial wastes, andbiological/chemical warfare agents is possible using an embodiment ofthe invention. Uploading of test results via radio transmission mayprovide remote sensing capabilities, and may provide responsecapabilities through human or central computer directed action. Responseinstructions may then be downloaded either to the sensing site or toanother strategic response position. Such a system may be useful, forexample, in determining the presence of toxins in a public water supply,and the subsequent centralized-directed cessation of water flow from thesupply pool.

[0465] In one embodiment, data may be collected from a remote locationand the data transmitted to a third party at an alternate location. Asample may be provided to a replaceable sensor cartridge having multipleanalyte sensors and being configured as part of the testing device. Thesample may be from a subject (e.g. a patient) and provided to thereplaceable sensor cartridge by an operator. Data regarding the sample,from the multiple analyte sensor, may be transmitted to a central dataservice. At the central data service, one or more tests may be performedon the electronic data using the central data service. After the testshave been performed, an electronic message may be transmitted to a thirdparty, remote from the central data service. The information may includethe results of one or more of the tests. In some embodiments, the testsmay be selected by the third party. After the third party has receivedthe results of the test the appropriate response (e.g. treatment in thecase of medical diagnostics) may be selected.

[0466] A sensor array system may also be used for remote diagnosticscreening. In one embodiment, a medical practitioner may prescribe atreatment to a patient during a visit. The medical practitioner may alsowish to monitor the quantity of treatment for the patient. In oneembodiment, the patient may provide a sample to a remote analyte testingdevice. The results produced by the analyte testing device may betransmitted to a central data service center. The central data servicecenter may perform an analysis of the data and make recommendations tothe patient to modify, maintain or cease the current treatment. Thetreatment may be in the form of medication, or an applied medicalprocedure. For medication treatments, the central data center may alsonote if any other medications are present. If so, the central dataservice center may advise the patient and/or practitioner of possibleadverse drug interactions. Allergic reactions may also be detected andreported in this manner.

[0467] Office visits may also be scheduled using the sensor arraysystem. Data collected from a patients sample may be sent from thesensor array system to a central data service. The electronic test datamay be analyzed at the central data service. The results of the testsmay be transmitted to a medical practitioner. The medical practitioner,upon review of the test results may schedule an appointment for thepatient. The subject may be notified of the need for an office visitthrough the central data service. Alternatively, the medicalpractitioner may decide that an office visit is unnecessary, but wish toalter the treatment. The medical practitioner may, either directly orindirectly (through the central data service) inform the patient of thechange of treatment.

[0468] Diagnostic Uses of the Sensor Array System

[0469] One of the largest markets for the health care industry is thediagnostic market. The worldwide market for diagnostic products is inthe range of about $20 billion a year. Much of this market is driven bythe current managed health care environment. The importance ofdiagnostics in the reduction of health care costs have created a needfor early and less expensive diagnosis. Generally, an early and accuratediagnosis may lead to early prognosis, reduced unnecessary testing andsignificantly lower health costs. This is especially true for animalmanagement. Animal diagnosis tends to be less accurate because of thetypes of testing being used. Instead of performing detailed diagnostictesting of animals, many animal care workers tend to prepare preventivemixtures which include a number of drugs for a variety of potentialdiseases that the animals may or may not have. These mixtures areexpensive and may, in the case of antibiotics, promote antibioticresistant pathogens.

[0470] The previously described sensor array system may be used for awide variety of diagnostic testing for both animals and humans. Asdescribed before, the sensor array may include a variety of particlesthat are chemically sensitive to a variety of types of analytes. In oneembodiment, the particles may be composed of polymeric beads. Attachedto the polymeric beads may be at least one receptor. The receptors maybe chosen based on their binding ability with the analyte of interest.

[0471] The sensor array may be adapted for use with a variety of bodilyfluids. Blood and urine are the most commonly used bodily fluids fordiagnostic testing. Other body fluids such as saliva, sweat, mucus,semen, and milk may also be analyzed using a sensor array. The analysisof most bodily fluids will, typically, require a filtration of thematerial prior to analysis. For example, cellular material and proteinsmay need to be removed from the bodily fluids. As previously described,the incorporation of filters onto the sensor array platform, may allowthe use of a sensor array with blood samples. These filters may alsowork in a similar manner with other bodily fluids, especially urine.Alternatively, a filter may be attached to a sample input port of thesensor array system, allowing the filtration to take place as the sampleis introduced into the sensor array.

[0472] In one embodiment, a sensor array may be customized for use as animmunoassay diagnostic tool imnunoassays rely on the use of antibodiesor antigens for the detection of a component of interest. In nature,antibodies are produced by immune cells in response to a foreignsubstance (generally known as the “antigen”). The antibodies produced bythe immune cell in response to the antigen will typically bind only tothe antigen that elicited the response. These antibodies may becollected and used as receptors that are specific for the antigen thatwas introduced into the organism.

[0473] In many common diagnostic tests, antibodies are used to generatean antigen specific response. Generally, the antibodies are produced byinjecting an antigen into an animal (e.g., a mouse, chicken, rabbit, orgoat) and allowing the animal to have an immune response to the antigen.Once an animal has begun producing antibodies to the antigen, theantibodies may be removed from the animal's bodily fluids, typically ananimal's blood (the serum or plasma) or from the animal's milk.Techniques for producing an immune response to antigens in animals arewell known.

[0474] Once removed from the animal, the antibody may be coupled to apolymeric bead. The antibody may then acts as a receptor for the antigenthat was introduced into the animal. In this way, a variety ofchemically specific receptors may be produced and used for the formationof a chemically sensitive particle. Once coupled to a particle a numberof well known techniques may be used for the determination of thepresence of the antigen in a fluid sample. These techniques includeradioimmunoassay (RIA) and enzyme immunoassays such as enzyme-linkedimmunosorbent assay (ELISA). ELISA testing protocols are particularlysuited for the use of a solid support such as polymeric beads. The ELISAtest typically involves the adsorption of an antibody onto a solidsupport. The antigen is introduced and allowed to interact with theantibody. After the interaction is completed a chromogenic signalgenerating process is performed which creates an optically detectablesignal if the antigen is present. Alternatively, the antigen may bebound to the solid support and a signal generated if the antibody ispresent. Immunoassay techniques have been previously described, and arealso described in the following U.S. Pat. Nos. 3,843,696, 3,876,504,3,709,868, 3,856,469, and 4,567,149 all of which are incorporated byreference.

[0475] In one embodiment, an immunoassay sensor array may be used forthe diagnosis of bacterial infections in animals. Many animals sufferfrom a variety of bacterial, viral, parasitic, and/or fungal diseasesthat may be unmonitored or not specifically diagnosed by the animalscaretakers. Bacterial infections may be particularly troublesome sincebacterial infections tend to cause a number of different healthproblems, some of which effect the quality of products produced from theanimals. It is desirable for the animal caretakers to diagnosis andtreat such problems as quickly as possible. The testing of animals,especially animals such as chickens and cattle, may be difficult due tothe large number of individual animals in the flock or herd. Adiagnostic tool for animal testing should be easy to use, accurate,quick and inexpensive. Such a tool would allow better animal healthmanagement, especially for large collections of animals.

[0476] For example, mastitis is a common bacterial infection that occursin the udders of cows. The presence of the mastitis causing bacteria incows may render the milk produced by the cows unsuitable for sale. Oncedetected, the treatment will involve the use of a mixture antibioticsthat also renders the milk unusable for a period of days. This canresult in a tremendous financial loss for the owners of the cows,especially if the infection spreads to the other cows of the herd.

[0477] Current mastitis detection includes daily observation of the bulktank somatic cell count. The bulk tank represent the bulk milk collectedfrom many different cows from the herd. The somatic cell count is ameasure of any inflammatory blood cells present in the milk of the cow,thus it is a measure of any inflammatory process that may have affectedthe udder of the cow. The somatic cell count offers a method ofscreening for potential problems in both the herd and the individualcows, but a confirmation test is necessary for a definitive diagnosis ofmastitis. The confirmation tests typically involve culturing the milkand analyzing the milk for the particular strains of bacteria that causemastitis. This process can take from 1 to 2 days to complete. Meanwhile,the communal use of milking machines may cause the infection to spreadwithin the herd.

[0478] The sensor array system described herein may be used to improvethe diagnostic procedures for testing milk samples for cows. In oneembodiment, antibodies that are specific for the bacteria that causemastitis may be bound to the receptors. Immunoassays for the detectionof mastitis are described in U.S. Pat. No. 5,168,044 which isincorporated by reference. Using the testing protocols previouslydescribed, the sensor array system may be used to detect the presence ofmastitis causing bacteria in any of the bodily fluids of a cow. Theimmunoassay is typically faster, (i.e., completed in hours instead ofdays) and may allow rapid sampling of individual members of the herd. Ingeneral, the immunoassays are much more accurate than cell culturemethods, which tend to give false positive results.

[0479] Another advantage of using a sensor array system, is thatmultiple bacterial strains may be analyzed simultaneously. Cow milk, aswell as other bodily fluids, may include other bacteria that maypotentially cause health problems for the animal. For example, a varietyof gram-positive bacteria such as staphylococcus and streptococcus andgram-negative bacteria such as coliforms (e.g., E. Coli), Proteus andPsuedomonas may also be present in the fluid sample. Typically, mastitistests ignore these bacteria, or in some cases, may confuse the presenceof these bacteria for the mastitis causing bacteria. In one embodiment,a sensor array may include multiple particles, each particle including areceptor that is specific for a particular bacterial strain. In a singletest, all of the bacteria present in the animal may be detected. This isparticularly important for determining the proper treatment of theanimal. By identifying the strains of bacteria present in the animal'ssample the appropriate antibiotics may be chosen for a treatment. Thismay help to avoid the proliferation of antibiotic resistant pathogensdue to unnecessary use of antibiotics.

[0480] While described in detail for the detection of mastitis in cows,it should be understood that the above described method of detectingbacteria in bodily fluids may be applied to a variety of differentbacteria found in both animals and/or humans. The sensor array wouldonly need to be modified with respect to the type of antibodies (orantigen) that are used for the testing procedure.

[0481] Additionally, bacteria in soil and grain samples may also bedetected using an immunoassay procedure. In the case of soils andgrains, an extraction of these mediums with a suitable solvent may berequired prior to analysis. For example, a grain sample may be soaked inwater and the undissolved material filtered out before the water isanalyzed. The analysis of the water may then take place using any of theprocedures for fluid samples previously described and the presence ofbacteria in the grain (or soil) may be determined.

[0482] In one embodiment, the sensor array is used to detectMycobacterium tuberculosis, the causative agent of tuberculosis.Immunoassays for the detection of Mycobacterium tuberculosis aredescribed in U.S. Pat. No. 5,631,130 which is incorporated by reference.

[0483] Many animals also suffer from a variety of parasitic diseases.Parasitic diseases may, occasionally, appear in humans as well. Forexample, one of the most prevalent parasitic disease found in dogs isheartworms. Heartworms are caused by the D. immitis parasite. The earlydetection of the presence of this parasite in a dog is important. Ifcaught at an early stage, the parasite may be treated with the use ofdrugs before any permanent damage to the heart is caused. A number oftests may be used for the detection of a heartworm infection. Those thatare most applicable for the sensor array system are based onimmunoassays. One test, known as the indirect fluorescent antibody testis specific for antibodies produced by the dog against the heartwormmicrofilaria. Another test utilizes an ELISA based screening fordetecting circulating worm antigen. Both of these immunoassay tests havea high degree of specificity for the detection of heartworms.

[0484] The previously described sensor array may be adapted for thedetection of heartworms using either of these well know techniques.Additionally, other parasitic infections may be simultaneously analyzedfor by the use of the additional particles which include receptors forother types of parasitic infections, including protozoan infections.Alternatively, a mixture of particles that are specific for eitherparasites or bacteria may be incorporated into a single sensor arrayunit. Since the analytes for bacteria and parasites tend to be found inthe same bodily fluids (e.g., blood), the use of such a sensor arraywould allow the diagnosis of potential bacterial and parasitic diseasesfor an animal (or a human) to be simultaneously detected. While theabove test has been described with respect to a dog, it should beunderstood that the testing procedure would be applicable to otheranimals and humans.

[0485] Another source of disease in humans and animals is from viralinfections. For example, feline leukemia is a viral infection that,until recently, was the most common fatal disease of cats. The diseaseis primarily caused by the exposure of the cat to the feline leukemiavirus (FeLV). The feline leukemia virus may be detected usingimmunoassay techniques. Three major tests have been used to determinethe presence of FeLV. The blood ELISA test is the most accurate, andwill detect the presence of FeLV at any stage of infection. FeLV antigenmay be used as a receptor that binds FeLV. An older test is based on aindirect fluorescent antibody (IFA) test for antibodies that areproduced against FeLV. A third test is a tears/saliva ELISA test. TheIFA and tear/saliva ELISA tests are only accurate in the late stages ofthe disease. As described above, the attachment of the appropriateantibodies or antigens on the particle will allow any of these testingprocedures to be performed using the sensor array system.

[0486] The HIV virus and the hepatitis C virus (togovirus andcalicivirus) are examples of viruses that humans may be tested for.These viruses are most commonly detected using an ELISA testing method.The ELISA testing methods for HIV or hepatitis C look for antibodies inthe bodily fluids of the person being tested. The most commonly analyzedbodily fluids used for these tests are blood and saliva. The attachmentof the appropriate antigens on a particle will allow any of thesetesting procedures to be performed using the sensor array system. Anadvantage of the use of a sensor array for the detection of viruses inhumans, is that many other pathogens may be simultaneously analyzed for.For example, viral infections from other viruses (e.g., hepatitis A,hepatitis B, human herpesvirus-8, cytomegalovirus, varicella zostervirus, etc.) and other pathogens (e.g., Pneumocystis carinii, Toxoplasmagondii, Mycobacterium avium, Mycobacterium intracellulare, Treponemapallidum, etc.) may be detected simultaneously with HIV and/or hepatitisC by the use of multiple particles with the appropriate antibodies orantigens. Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium,Mycobacterium intracellulare, cytomegalovirus, human herpesvirus-8, andvaricella zoster virus are organisms that cause infections inimmunocompromised patients. Treponema pallidum is the bacteria thatcauses syphilis. Immunoassays for the detection of Pneumocystis cariniiare described in U.S. Pat. No. 4,925,800 which is incorporated byreference. Immunoassays for the detection of Toxoplasma gondii,cytomegalovirus, Herpes simplex virus, and Treponema pallidum aredescribed in U.S. Pat. No. 4,294,817 which is incorporated by reference.Immunoassays for the detection of Toxoplasma gondii are also describedin U.S. Pat. No. 5,965,590 which is incorporated by reference.Immunoassays for the detection of Hepatitis B use Hepatitis B surfaceantigen as a receptor.

[0487] It should be understood that parasitic, viral and bacterialinfections may all be analyzed at substantially the same time using thesensor array system. The sensor array system may include all of thenecessary reagents and indicators required for the visualization of eachof these tests. In addition, the sensor array may be formed such thatthese reagents are compartmentalized. In this manner, the reagentsrequired for a viral tests may be isolated from those used for abacterial test. The sensor array may offer a complete pathogen analysisof an animal or persons bodily fluid with a single test.

[0488] The presence of fungus in grains may also be detected using asensor array system. The fungus in grains may be removed using anextraction technique. The samples may be analyzed with a sensor arraysystem which includes particles that are sensitive to the presence of avariety of fungi. In this was, the fungi present in a grain sample maybe monitored.

[0489] Diagnostic tests have also been used for the detection of variousorganic molecules in humans and animals. These molecules may be detectedby a variety of testing procedures, including, but not limited to,immunoassay techniques, enzyme binding techniques, and syntheticreceptors.

[0490] The concentration of glucose in human blood is commonly measuredfor people with diabetes. The measurement of the blood glucose level maybe performed more than 5 times a day for some individuals. Currentlyavailable home testing relies, primarily, on a blood test for thedetermination of the concentration of glucose. The determination ofglucose is typically determined by the enzymatic decomposition ofglucose. Some methods for the determination of glucose in blood aredescribed in U.S. Pat. Nos. 3,964,974 and 5,563,042 which areincorporated by reference.

[0491] Cholesterol is also a common constituent of blood that isfrequently monitored by people. As with glucose, a number of hometesting kits have been developed that rely on the use of an enzyme basedtesting method for the determination of the amount of cholesterol inblood. A method for the determination of cholesterol in blood isdescribed in U.S. Pat. No. 4,378,429 which is incorporated by reference.

[0492] The triglyceride level in blood is also commonly tested forbecause it is an indicator of obesity, diabetes, and heart disease. Asystem for assaying for triglycerides in bodily fluids is described inU.S. Pat. No. 4,245,041 which is incorporated by reference.

[0493] The concentration of homocysteine may be an important indicatorof cardiovascular disease and various other diseases and disorders.Various tests have been constructed to measure the concentration ofhomocysteine in bodily fluids. A method for the determination ofhomocysteine in blood, plasma, and urine is described in U.S. Pat. No.6,063,581 which is incorporated by reference.

[0494] Cholesterol, triglyceride, homocysteine, and glucose testing maybe performed simultaneously using the sensor array system. Particlesthat are sensitive to either cholesterol, triglyceride, homocysteine, orglucose may be placed in the sensor array. Blood serum that is passedover the area may, therefore, be analyzed for glucose, triglyceride, andcholesterol. A key feature of a glucose, triglyceride, homocysteine,and/or cholesterol test is that the test should be able to reveal theconcentration of these compounds in the persons blood. This may beaccomplished using the sensor array by calibrating the reaction of theparticles to cholesterol, triglyceride, or glucose. The intensity of thesignal may be directly correlated to the concentration. In anotherembodiment, multiple particles may be used to detect, for example,glucose. Each of the particles may be configured to produce a signalwhen a specific amount of glucose is present. If the glucose present isbelow a predetermined concentration, the particle may not produce adetectable signal. By visually noting which of the particles areproducing signals and which are not, a semi-quantitative measure of theconcentration of glucose may be determined. A similar methodology may beused for cholesterol, triglyceride, homocysteine, or any testing systemthereof (e.g., glucose/cholesterol/triglyceride/homocysteine,cholesterol/triglyceride, glucose/triglyceride, glucose/cholesterol,etc.).

[0495] Another use for the sensor array system is in hormone testing.The most common types of hormone testing in use today are fertilitytesting devices (e.g., pregnancy tests and ovulation tests). Both ofthese tests typically rely on either an immunoassay or enzyme assaymethodology. Other hormones, such as progesterone for fertilitymonitoring or estrogen for hormone therapy treatments may also bemonitored. Th sensor array may be used in hormone testing for specifichormones or for multiple hormones in a manner similar to that describedfor glucose/cholesterol testing.

[0496] Another practical use for the sensor array system is fortherapeutic drug monitoring. Therapeutic drug monitoring is themeasurement of the serum level of a drug and the coordination of thisserum level with a serum therapeutic range. The serum therapeutic rangeis the concentration range where the drug has been shown to beefficacious without causing toxic effects in most people. Typically,therapeutic drug monitoring relies on the analysis of blood serum orplasma from a patient. In general, therapeutic drug monitoring relies onthe use of immunoassays, similar to the ones described previously.

[0497] A general problem with monitoring of drug serum levels may occurwhen a patient is using more than one drug. In some instances, the drugsmay produce a positive result in an immunoassay, especially if the drugshave a similar chemical structure. In some instances, the receptor(antibody or antigen) may be altered to prevent a particularinterference. The use of a sensor array, however, may avoid thisproblem. Because a sensor array may include a variety of differentparticles, each of the particles may be customized for a particulardrug. If multiple drugs are present in a patients serum, the presence ofthe drugs may be determined by observing which of the particles isactivated. Even though some of the particles may be reactive to morethan one of the drugs, other receptors may be more finely tuned to aspecific drug. The pattern and intensity of the reactions of theparticles with the drugs may be used to accurately assess the drugspresent in the patient.

[0498] One area of therapeutic monitoring includes the monitoring ofanticonvulsant drugs. Anticonvulsant drugs are usually measured by animmunoassay. Common anticonvulsant drugs that require monitoring includephenytoin (Dilantin®), carbamazepine (Tegretol®), valproic acid(Depakene®), primidone (Mysoline®), and phenobarbital. Since primidoneis metabolized to phenobarbital, both drugs must be measured when thepatient is taking primidone.

[0499] Another of therapeutic drug monitoring that a sensor array may beused for is the monitoring of Digoxin. Digoxin is a medicine that slowsthe heart and helps it pump more effectively. The bioavailability ofdifferent oral preparations of digoxin tends to be highly variable frompatient to patient. Digoxin measurements may be made using animmunoassay. Some immunoassays for digoxin, however, havecross-reactivity with a hormone-like substance know as digoxin-likeimmunoreactive factor, or DLIF. Care must be taken to distinguishbetween digoxin and digitoxin, another cardiac glycoside. Digoxin assaysgenerally have a low cross-reactivity with digitoxin, but digitoxinserum therapeutic levels may be 10 times those of digoxin. The use of asensor array system that includes a variety of particles, some of whichare more sensitive to DLIF or digitoxin, may allow a more accurateassessment of digoxin levels in a patient.

[0500] Theophylline is a bronchodilator with highly variableinter-individual pharmacokinetics. Serum levels must be monitored afterachievement of steady-state concentrations to insure maximum therapeuticefficacy and prevent toxicity. Immunoassay is the most common methodused for monitoring this drug.

[0501] Lithium compounds are used to treat bipolar depressive disorders.Serum lithium concentrations are measured by ion selective electrodetechnology. An ion selective electrode has a membrane which allowspassage of the ion of interest but not other ions. A lithium electrodewill respond to lithium concentrations but not to other small cationssuch as potassium. Several small analyzers which measure lithium usingion selective electrode technology are available. The use of particlesthat are sensitive to lithium ion concentrations, as have been describedpreviously, may allow lithium ion measurements to be preformed withoutthe use of lithium ion electrodes. Such systems will allow the analysisof multiple ions in the serum, unlike the electrode based systems whichare specific for lithium ions.

[0502] The tricyclic antidepressant drugs include imipramine and itspharmacologically active metabolite desipramine; amitriptyline and itsmetabolite nortriptyline; and doxepin and its metabolite nordoxepin.Both the parent drugs and the metabolites are available aspharmaceuticals. These drugs are primarily used to treat bipolardepressive disorders. Imipramine may also be used to treat enuresis inchildren, and severe Attention Deficit Hyperactivity Disorder that isrefractory to methylphenidate. Potential cardiotoxicity is the majorreason to measure these drugs. Immunoassay methods are available formeasuring imipramine and the other tricyclics. When measuring tricyclicantidepressants which have pharmacologically active metabolites, it isimportant to measure both the parent drug and the metabolite. A sensorarray system is well suited for this type of analysis. Mixtures ofreceptors for parent drug and the metabolites may be incorporated into asingle sensor array. In addition, a sensor array system may be used todetect a variety of tricyclic antidepressant drugs, allowing any of thedrugs to be tested using a single test.

[0503] Screening patients for drugs of abuse in the urine may beindicated to help differentiate symptoms, or to insure that a patient issubstance-free before undergoing medical procedures. Drug screening ofpregnant women with a history of drug abuse may be useful as aneducational tool and help guide treatment of the newborn. In addition,some employers require a drug screen as part of an employment orpre-employment physical. Nearly all workers in some occupations, such aslaw enforcement and transportation, are subject to periodic, random, andpost-incident drug screening. The chemical sensor array may be used todetect a variety of drugs of abuse in a quick and easy manner.Typically, a variety of different tests must be used to test for eachclass of drug. By incorporating multiple particles into a single sensorarray, some or all of the most commonly used drugs of abuse may bedetermined in a single step.

[0504] Urine screening tests for drugs of abuse detect general classesof compounds, such as amphetamines, barbiturates, benzodiazepines, oropiates. Drug screening also includes testing for cocaine, marijuana,and phencyclidine (PCP). The screening test for cocaine detects benzoylecgonine, the major metabolite of cocaine. The marijuana test detectsD-9-tetrahydrocannabinol, a principle product of marijuana smoke. Oneproblem of the screening test is that the test, in some instances, maynot be able to distinguish between illicit drugs and prescription orover-the-counter compounds of the same class. A patient taking codeineand another taking heroin would both have a positive screening test foropiates. Some over-the-counter medications can cause a positive drugscreen in a person who has not taken any illegal or prescription drugs.For instance, over-the-counter sympathomimetic amines such aspseudoephedrine and phenylpropanolamine may cause a false-positivescreen for amphetamines. Eating food containing poppyseeds may result ina positive urine screening test for opiates, since poppyseeds containnaturally-occurring opiates. However, confirmation testing willdistinguish between positive opiate tests resulting from poppyseedingestion and those resulting from heroin or other opiates, becausedifferent metabolic breakdown products are present. Monoacetylmorphine(also called 6-monoacetylmorphine or 6-MAM) is a heroin metabolite. Thepresence of this metabolite is conclusive evidence that heroin wasingested.

[0505] Most of these problems of false positive results may be avoidedthrough the use of a sensor array. The sensor array may include avariety of particles, each specific for a particular drug. Some of theparticles may be specific designed to interact with the drug of abuse,for example amphetamine. Other particles may be designed to interactwith an over-the-counter drug such as pseudoephedrine. The use of avariety of particles may allow a more accurate or complicated analysisto be performed through the use of a pattern recognition system. Eventhough many of the drugs may react with one or more particles, thepattern and intensity of the signals produced by the particles in thesensor array may be used to determine the identity of the drugs presentin the patient. The most commonly used test method for screening urinefor drugs of abuse is immunoassay. A number of single use devicesincorporating immunoassays and designed to be used outside of thetraditional laboratory are currently available.

[0506] Hyperglycemia can be diagnosed only after ruling out spuriousinfluences, especially drugs, including caffeine, corticosteroids,indomethacin, oral contraceptives, lithium, phenytoin, furosemide,thiazides, etc. Thus, a sensor array may be used to expedite diagnosisof hyperglycemia by determining the presence of drugs that may causefalse positives.

[0507] In another embodiment, a sensor array may be used to asses thepresence of toxins in a person or animal's system. In general, toxinsmay be any substance that could be ingested that would be detrimental toone's health. For animals, a few examples of toxins include lead,organic phosphates, chlorinated hydrocarbons, petroleum distillates,alkaloids (present in many types of poisonous plants), ethylene glycol,etc. People may ingest a variety of these compounds, along with a numberof different types of drugs, either over-the counter, prescription, orillegal. In many instances the patient, either animal or human, mayexhibit symptoms which indicate the presence of a poison, however, thediagnosis of the particular poison ingested by the person may bedifficult. This may be particularly difficult for animals or children,since the owner may not know what the animal/child has eaten. Forpeople, if the poisoning is severe, the person may be unconscious andunable to tell the physician the cause of the poisoning.

[0508] The use of a sensor array, may allow a medical expert toaccurately and quickly assess the types of toxins present in a patient.A single sensor array may hold particles that are reactive to a widevariety of toxins. A single analysis of a sample of the patients bodilyfluids (e.g., blood) may allow the medical expert to determine theidentity of the poison. Once identified, the proper treatment may beused to help the patient.

[0509] A sensor array may also be used for soil testing. As with thegrain testing, the testing of soil samples may require an extraction ofthe soil samples by a suitable solvent. For metals and other inorganicsalts, the solvent used may be either water or dilute aqueous acidsolutions. The soil may also be extracted with organic solvents toextract any organic compounds that are present in the soil sample. Thesesolution containing the extractable material may then be analyzed usinga sensor array. The sensor array may include particles that are specificfor a variety of soil contaminates such as paints, lead, phosphates,pesticides, petroleum products, industrial fallout, heavy metals, etc.The use of a sensor array may allow one or more of these materials to besimultaneously analyzed in a soil sample.

EXAMPLES

[0510] In the below recited table are examples of analytes that havebeen detected using the sensor array system described herein. In theReceptor/Enzyme column are listed examples of receptors that may be usedfor the corresponding analyte. These receptors are covalently bound to apolymeric resin, using methods described herein. Analyte TypeReceptor/Enzyme Sodium, Potassium Small Molecule (Electrolyte) Crownethers, cryptands, chromoionophores such as Chromolyte ® (from Bayer),Enzymes such as β- galactosidase, or other metalloenzymes. BicarbonateSmall Moleculae (Electrolyte) Enzymes such as Carbonic anhydrase CalciumSmall Molecule (Electrolyte) Complexometric dyes such as Arsenazo III,Zylneol Orange, Alizaren Complexone Magnesium Small Molecule(Electrolyte) Complexometric dyes such as Calmagite, Magon ChlorideSmall Molecule (Electrolyte) Enzymes and/or small molecule detectorssuch as Amylase, Phenyl mercury compounds, mercuric thiocyanates,diphenylcarbazones Oxygen Small Molecule (Metabolite) Oxygen complexingmolecules such as porphyrins, synthetic hemeglobins, Rutheniumtribipyridine Carbon dioxide Small Molecule (Metabolite) Enzymes such asCarbonic anhydrase pH Small Molecule (Electrolyte) PH indicator dyessuch as Hydroxynitrophenylacetic acid, Congo Red, Brilliant Yellow,Carboxyphenolphthalein Creatinine Small Molecular (Metabolite) Enzymessuch as Creatinine deiminase or small molecule detectors such as picrateUrea Small Molecule (Metabolite) Enzymes such as Urease Glucose SmallMolecule (Metabolite) Enzymes such as Gluocose oxidase/PeroxidaseHepatitis B Virus Antigen/antibody pairs such as Hepatitis B surfaceantigen Feline Leukemia Virus Antigen/antibody pairs such as FeLVantigen Cytokines Interleukin Small Molecule (Markers), Small moleculemarkers and/or 1 Interleukin 2 Cellular signals antigen/antibody pairsInterleukin 4 Interleukin 6 Interleukin 10 Gamma Interferon TumorMecrosis Factor (TNF)

[0511] Nucleic Acid Identification Methodology

[0512] In one embodiment, the chemical sensor array may be used for thedetermination of the sequence of nucleic acids. Generally, a receptormay be attached to a polymeric bead to form a particle. The receptor mayhave a specificity for a predetermined sequence of a nucleic acid.Examples of receptors include deoxyribonucleic acids (DNA) natural orsynthetic (e.g., oligomeric DNA), ribonucleic acids (RNA) natural orsynthetic, and enzymes. A number of methods may be used to analyze anucleic acid to determine its sequence. The methods, summarized below,may be adapted for use in the previously described chemical sensor arrayto analyze a sample which includes a nucleic acid analyte.

[0513] In one embodiment, hybridization may be used to identify nucleicacids. This method relies on the purine-pyrimiidine pairing propertiesof the nucleic acid complementary strands in the DNA-DNA, DNA-RNA andRNA-RNA duplexes. The two strands of DNA are paired by the establishmentof hydrogen bonds between the adenine-thymine (A-T) bases and theguanine-cystosine (G-C) bases. Hydrogen bonds also form theadenine-uracil (A-U) base pairs in the DNA-RNA or RNA-RNA duplexes.Hybridization is highly sequence dependent. Sequences have the greatestaffinity with each other where, for every purine in one sequence(nucleic acid) there exists a corresponding pyrimidine in the othernucleic acid and vice versa. The target fragment with the sequence ofinterest is hybridized, generally under highly stringent conditions thattolerate no mismatches. U.S. Pat. No. 6,013,440 to Lipshutz, et al.describes hybridization in further detail and is incorporated byreference as if fully set forth herein.

[0514] Despite the high specificity of hybridization, there may be somemismatched nucleic acid strands. There are several ways to preventmismatched strands from causing false positives. Ribonuclease enzymesmay be used to dispose of mismatched nucleic acid pairs forming aRNA/DNA or RNA/RNA hybrid duplex. There are many types of ribonucleaseenzymes that may be used for this purpose, including RNase A, RNase T1and RNase T2. Ribonuclease enzymes specifically digest single strandedRNA. When RNA is annealed to form double stranded RNA or an RNA/DNAduplex, it may no longer be digested with these enzymes. When a mismatchis present in the double stranded molecule, however, cleavage at thepoint of mismatch may occur. In one embodiment, a label may be attachedto the RNA coupled to the particle. In the presence of a mismatch,cleavage may occur at the point of the mismatch. The cleavage may causethe labeled fragment to fall off the bead, causing a decrease in thesignal detected from the bead. If the nucleic acid are perfectlycomplementary, then the fragment may remain uncleaved in the presence ofthe ribonuclease enzymes and the intensity of the signal produced by theparticle may remain unchanged.

[0515] S1 Nuclease Cleavage may also be used to cleave mismatched pairs.S1 nuclease, an endonuclease specific for single-stranded nucleic acids,may recognize and cleave limited regions of mismatched base pairs inDNA:DNA or DNA:RNA duplexes. Normally, for S1 Nuclease to recognize andcleave a duplex a mismatch of at least about four consecutive base pairsis required. In a similar manner as described above, the cleavage of alabeled nucleic acid fragment may indicate the presence of a mismatchednucleic acid duplex.

[0516] T4 endonuclease VII (T4E7) and T7E1 are small proteins frombacteriophages that bind as homodimers and cleave aberrant DNAstructures including Holliday Junctions. These molecules preferentiallycleave mismatched duplexes. (Described in Youil R, Kemper B, Cotton RGH.Detection of 81 of 81 Known Mouse Beta-Globin Promoter Mutations With T4Endonuclease-VII—The EMC Method. Genomics 1996;32:431-5, incorporated byreference as if fully set forth herein).

[0517] In another method, Chemical Cleavage of Mismatches (CCM) may beused. This technique relies upon the use of intercalation. Examples ofintercalators include, but are not limited to, the chemicalshydroxylamine and osmium tetroxide to react with a mismatch in a DNAheteroduplex. Mismatched thymines are susceptible to modification byosmium tetroxide (or tetraethyl ammonium acetate and potassiumpermanganate) and mismatched cytosines can be modified by hydroxylamine.The modified bases are then cleaved by hot piperidine treatment. In asimilar manner as described above, the cleavage of a labeled nucleicacid fragment may indicate the presence of a mismatched nucleic acidduplex.

[0518] In another embodiment, DNA-binding proteins may be used toidentify nucleic acids. Most sequence-specific DNA-binding proteins bindto the DNA double helix by inserting an a-helix into the major groove(Pabo & Sauer 1992 Annu. Rev. Biochem. 61. 1053-1095; Harrison 1991Nature (London) 353, 715-719; and Klug 1993 Gene 135, 83-92). U.S. Pat.No. 5,869,241 to Edwards, et al. describes in detail methods foridentifying proteins having the ability to bind defined nucleic acidsequences and is incorporated by reference as if fully set forth herein.In an embodiment, the DNA-binding proteins may be attached to apolymeric particle. The DNA-binding proteins may interact with thepolymeric particle to produce a signal using a variety of the previouslydescribed signaling protocols.

[0519] Mispair Recognition Proteins, e.g., MutS, may also be used todetect mismatched base pairs in double-stranded DNA. There are severalmethods by which Mispair Recognition Proteins can be used. MispairRecognition Proteins may bind to a mismatched base pair. Modified formsof a mismatch recognition protein may cleave a heteroduplex in thevicinity of a mismatched pair. A mismatch repair system dependentreaction, e.g., MutBLS, may be used for mismatch-provoked cleavage atone or more GATC sites. A mismatch repair system may be used in theformation of a mismatch-provoked gap in heteroduplex DNA.Mismatch-containing nucleotides may be labeled with a nucleotide analog,e.g., a biotinylated nucleotide. Molecules containing a base pairmismatch may be removed through the binding of the mismatch to thecomponents of the mismatch repair system or by the binding of a complexof a mismatch and components of a mismatch repair system to othercellular proteins. Molecules containing mismatches may also be removedthrough the incorporation of biotin into such a molecule and subsequentremoval by binding to avidin. The use of Mispair Recognition Proteins isdescribed in detail in U.S. Pat. No. 6,008,031 to Modrich, et al., whichis incorporated by reference as if fully set forth herein. Hsu I C, YangQ P, Kahng M W, Xu J F. Detection of DNA point mutations with DNAmismatch repair enzymes. Carcinogenesis 1994;15: 1657-62.1, which isincorporated by reference as if fully set forth herein, describes theuse of MutY in combination with thymine glycosylase for mismatchdetection.

[0520] Yet another technique is Oligonucleotide Ligation Assay. In thismethod, the enzyme DNA ligase is used to join two oligonucleotides,annealed to a strand of DNA, that are exactly juxtaposed. A single basepair mismatch at the junction of the two oligonucleotides will preventligation. Ligation is scored by assaying for labels on the twooligonucleotides becoming present on a single molecule.

[0521] In another embodiment, an intercalating molecule may be used as areceptor. The combination of the intercalator with the polymeric beadmay be used as a particle for a sensor array system. Intercalatorstypically react with duplex DNA by insertion into the duplex DNA. If theintercalator has a visible or ultraviolet absorbance or fluorescence,the wavelength or intensity of the intercalators signal may be alteredwhen the intercalator is intercalated into duplex DNA. Examples of suchintercalators include, but are not limited to, ethidium bromide, POTO,and Texas Red. Many intercalators exhibit some sequence selectivity.Thus, an intercalator bound to a polymeric resin may be used toanalyzing DNA analytes for specific sequences. By using a variety ofdifferent intercalators in a single sensor array, the identity of thenucleic acid may be identified through a pattern recognitionmethodology.

[0522] The use of particles that are custom made for a variety ofdifferent nucleic acid testing schemes allows greater flexibility thanthe current commercially available nucleic acid devices. For example,the use of silicon chips in which the nucleic acid receptor is coupleddirectly to the chip may be less flexible since the size of theoligomeric receptor built onto the chip is limited to 25-30 base pairs.Methods for synthesizing oligomeric nucleic acids on a bead, however,may be used to couple oligomeric nucleic acids which include more than100 base pairs.

[0523] Tests used to identify nucleic acids sometimes require that theamount of nucleic acid in the sample be increased. Techniques have beendeveloped to amplify the chemical of interest. For example, it ispossible to control which strand of a duplex nucleic acid is amplifiedby using unequal amounts of primer so that the primer for the undesiredstrand is effectively rate limiting during the amplification step.Methods of determining appropriate primer ratios and template sense arewell known to those of skill in the art (see, e.g., PCR Protocols: aGuide to Methods and Applications, Innis et al., eds. Academic Press,Inc. N.Y. 1990).

[0524] Polymerase Chain Reaction (PCR) is a widely used technique whichenables a scientist to amplify DNA and RNA sequences at a specificregion of a genome by more than a millionfold, provided that at leastpart of its nucleotide sequence is already known. The portions on bothsides of the region to be amplified are used to create two synthetic DNAoligonucleotides, one complementary to each strand of the DNA doublehelix, which serve as primers for a series of synthetic reactions whichare catalyzed by a DNA polymerase enzyme. Effective amplification mayrequire up to 30 to 40 repetitive cycles of template nucleic aciddenaturation, primer annealing and extension of the annealed primers bythe action of a thermostable polymerase. A more detailed description aswell as applications of PCR are provided in U.S. Pat. Nos. 4,683,195;4,683,202; and 4,965,188; Saiki et al., 1985, Science 230:1350-1354;Mullis et al., 1986, Cold Springs Harbor Symp. Quant. Biol. 51:263-273;Mullis and Faloona, 1987, Methods Enzymol. 155:335-350; PCRTechnology-principles and applications for DNA amplification, 1989, (ed.H. A. Erlich) Stockton Press, New York; PCR Protocols: A guide tomethods and applications, 1990, (ed. M. A. Innis et al.) Academic Press,San Diego; and PCR Strategies, 1995, (ed. M. A. Innis et al.) AcademicPress, San Diego, Barany, 1991, PCR Methods and Applic. 1:5-16); Gap-LCR(PCT Pat. Publication No. WO 90/01069); each of which is incorporated byreference as if fully set forth herein.

[0525] In Allele-Specific PCR (also called the amplification refractorymutation system or ARMS) the assay occurs within the PCR reactionitself. Sequence-specific PCR primers which differ from each other attheir terminal 3′ nucleotide are used to only amplify the normal allelein one reaction, and only the mutant allele in another reaction. Whenthe 3′ end of a specific primer is fully matched, amplification occurs.When the 3′ end of a specific primer is mismatched, amplification failsto occur.

[0526] Other amplification techniques include Ligase Chain Reaction,described in Wu and Wallace, 1989, Genomics 4:560-569 and Barany, 1991,Proc. Natl. Acad. Sci. USA 88:189-193, incorporated by reference as iffully set forth herein; Strand Displacement Amplification; Nucleic AcidSequence Base Amplification; Transcription Mediated Amplification;Repair Chain Reaction, described in European Pat. Publication No.439,182 A2), 3SR (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878; PCT Pat. Publication No. WO 92/0880A), and NASBA (U.S.Pat. No. 5,130,238), incorporated by reference as if fully set forthherein; Self-sustained Sequence Replication; Strand DisplacementAmplification, etc., described in Manak, DNA Probes, 2^(nd) Edition, p255-291, Stockton Press (1993)), incorporated by reference as if fullyset forth herein; and Non-Isotopic RNase Cleavage Assay, described inGoldrick M M, Kimball G R, Liu Q, Martin L A, Sommer S S, Tseng J Y H.Nirca(Tm)—A Rapid Robust Method For Screening For Unknown PointMutations. Biotechniques 1996;21:106-12, incorporated by reference as iffully set forth herein. Non-Isotopic RNase Cleavage Assay amplifies RNA.RNase enzymes, e.g., RNase 1 and RNase T1, increase the sensitivity ofthe assay.

[0527] Manufacturing Methods for a Sensor Array

[0528] As described above, after the cavities are formed in thesupporting member, a particle may be positioned at the bottom of acavity using a micromanipulator. This allows the location of aparticular particle to be precisely controlled during the production ofthe array. The use of a micromanipulator may, however, be impracticalfor mass-production of sensor arrays. A number of methods for insertingparticles that may be amenable to an industrial application have beendevised.

[0529] In one embodiment, the use of a micromanipulator may beautomated. Particles may be “picked and placed” using a roboticautomated assembly. The robotic assembly may include one or moredispenser heads. A dispenser head may be configured to pick up and holda particle. Alternatively, a dispenser head may be configured to hold aplurality of particles and dispensing only a portion of the heldparticles. An advantage of using a dispense head is that individualparticles or small groups of particles may be placed at preciselocations on the sensor array. A variety of different types of dispenseheads may be used.

[0530] In one embodiment, a vacuum pick-up/dispense head may be used.The dispense head uses a vacuum system to pick up particles. Thedispense head may be formed using small diameter tubing, with an innerdiameter (ID) smaller than the particle outer diameter (OD). Thedispense head may be coupled to a robotic control system via an arm. Therobotic control system may be programmed to first move the dispense headto a storage location of the correct particle type, vacuum would beapplied to the dispense head once it is “dipped” into the particlestorage compartment, thus grasping one particle. The robotic controlsystem would then move the arm such that the dispense head is in aposition in close proximity to (or actual contact with) the appropriatelocation on the sensor array (See FIG. 70A). The dispense head vacuumwould then be turned off (i.e., the vacuum would be removed), and ifnecessary a slight positive pressure could be applied to the dispensehead. The particle would thus be dislodged from the dispense head ontothe sensor array (See FIG. 70B).

[0531] The robotic control system may include a single dispense head ora plurality of dispense heads. The use of a plurality of dispense headswould allow multiple cavities of the sensor array to be filled during asingle filing operation. In this manner the efficiency of filling thesensor array may be increased.

[0532] Another example of a robotic vacuum pick-up/dispense head isdescribed in U.S. Pat. No. 6,151,973 to Geysen which is incorporatedherein by reference.

[0533] The dispense head could also be in the form of a “solid” pick-upwand. The solid dispense head may rely on natural attractive forcesbetween a particle and the dispense head material to attach a particleto the dispense head. For example, when a particle is placed in closeproximity to the dispense head, electrostatic interactions between theparticle and the dispense head may cause the particle to “stick” to thedispense head. The dispense head may be placed at the appropriatelocation over a cavity of the sensor array (See FIG. 71A). When theparticle is placed in close proximity to the sensor array, theattractive forces between the chip and particle, along withgravitational forces, may cause the particle to transfer from thedispense head to the sensor array (See FIG. 71B). For example, with PEGparticles, a dispense head made of tungsten will cause the PEG particleto attach to the tungsten tip, but the particle may still be transferredto a silicon based sensor array when brought into close proximity of thesensor array. A single solid dispense head or a plurality of soliddispense heads may be used.

[0534] In another embodiment, the dispense head could also be formedfrom one or more “pipettes” with an inner diameter greater than thediameter of the particles. Particles may be delivered directly into thebore of the pipette using a pump/dispense system. Such a system issimilar to precision adhesive dispense systems in current use. Theparticles may be suspended in a liquid (e.g., water), and controlledamounts of the liquid would be pumped through the head to deliver aparticle to the appropriate location on the sensor array chip. Such adispensing system may have difficulties delivering only one particle ata time. Any extra particles, however, may be removed form the sensorarray after application. Additionally, by making an array of pipettesthe rate of particle placement may be increased. Other advantages ofthis approach may include the ability to deliver the particles in anaqueous environment if the particle chemistry so requires, as wellmaking the deliver of different particles to each head fast andefficient, since no “pick up” step is required.

[0535] The “pipette” system relies on the use of controlled amounts ofliquid to transport the particle from a storage area to the tip of thedispense head. In one embodiment, blast of air may be used to force aportion of the liquid toward the dispense head tip. In anotherembodiment, the dispense head may be made using technology essentiallyidentical to that used in “ink-jet” printer heads. These heads typicallyrely on bursts of heat to quickly heat the liquid, causing bubbles ofthe liquid to be forced to the tip of the dispense head.

[0536] Once the pick-up/dispense head has delivered a particle orcollection of particles to the appropriate location on the sensor arrayit may be desirable to insure that a single particle be collected atexactly the correct position on the sensor array. This may beaccomplished using a vacuum chuck-like effect, as illustrated in FIGS.72A-72D.

[0537] In one embodiment, the sensor array includes cavities used tolocate and at least partially contain the particles. When placed on amain vacuum chuck, each individual cavity may also acts as a vacuumchuck. The sensor array, when placed on a vacuum chuck may allowair-flow through the cavities. FIG. 72A depicts a multi-tip dispensehead that allows the simultaneous application of many particles. Thehead is aligned to the cavities in the sensor array using an appropriatemechanical alignment system. If a particle is simply brought intoproximity with the cavity, the fluid (e.g., air, but could also be aliquid) flow through the cavity may draw the particle into its properlocation and hold it there (as depicted in FIG. 72B). In someembodiments, the dispense head may delivered more than one particle to agiven cavity. Only one of the dispensed particles, however, is fullyheld in place by the vacuum at any give cavity. After the dispense headis moved away from the sensor array, excess particles may be removedusing a side-directed jet (air or some other fluid) as depicted in FIG.72C. The desired particles are held in their storage pits by thepressure differential across pits produced by the vacuum chuck. Theprocess may now be repeated. In FIG. 72D another pipette head isillustrated that dispenses a distinct set of particles from thatdispensed by the first head. This may allow more rapid dispensing of alarger variety of particle types.

[0538] When the sensor array is placed on a vacuum chuck, the particlesmay be picked up with a vacuum dispense head. The particles may then bepulled off of the dispense tool when the vacuum of the dispense head isreleased. The applied vacuum from the vacuum chuck may keep theparticles from in the cavities. After the particles have been dispensed,a cover may be disposed on the sensor array to keep the particles inplace. The cover may be attached to the sensor array using a pressuresensitive adhesive. After the cover is placed onto the sensor array, thevacuum may be released and the sensor array removed from the vacuumchuck.

[0539] Passive Transport of Fluid Samples

[0540] For some chemical sensor array systems, fluids may be transportedinto and across the sensor array during use. In one embodiment, fluidsmy be transferred into and through a sensor array using a system thatrelies on variations in the surface wetting characteristics of achannel. An advantage of such a system is that the system may be“passive” (i.e., no external power source or components). Upon theintroduction of a sample, the samples may be drawn into the system anddistributed to the particles. This is particularly advantageous forsmall portable sensor array systems.

[0541] In one embodiment, a chemical sensor array is composed of anumber of superimposed layers. FIG. 73 depicts a side-sectional view ofthe sensor array system. A support layer 1010 (e.g., a glass layer) isused as the foundation for the system. A spacer layer 1020 is formedupon the support layer. The support layer may be formed of a relativelyinert material using standard semiconductor lithographic techniques. Inone embodiment, the support layer may be formed from photoresist (e.g.,a dry film photoresist). Alternatively, silicon nitride or silicondioxide may be used as the spacer layer. The spacer layer may bepatterned such that the spacer layer supports an outer portion of anoverlying sensor layer 1030. This etching of the spacer layer 1020 mayform a channel 1022 under the cavities formed in the sensor layer 1030.This channel 1022, may allow fluids to pass through the cavities and outof the sensor array system.

[0542] The sensor layer 1030 includes a number of cavities 1036 forholding a particle 1038. The formation of cavities in a sensor layer hasbeen described earlier. In one embodiment, the sensor layer is formedfrom silicon. The silicon sensor layer may be partially etched such thatan inlet and channel may be formed in the silicon layer. As depicted inFIG. 73 the outer portion of the sensor layer may be thicker than theinterior portions. The application of a cover layer 1050, may beaccomplished by resting the cover layer on the elevated portions of thesensor layer. This creates a channel 1042 between the cover layer andthe sensor layer.

[0543] The etched portion of the sensor layer may be divided intosegments coupled by a channel. FIG. 74A depicts a top view of the sensorarray system and FIG. 74B depicts a bottom view. The first segment 1041acts as a well or reservoir for the introduction of fluid samples. Thesecond segment 1045 may include a number of cavities which includeparticles. The first segment may be coupled to the second segment by oneor more channels 1043, formed in the sensor array. The channels allowthe fluid to flow from the reservoir to the cavities. The cover layer1050 may be positioned over the support layer 1030 to form channel 1042.Materials and methods of for forming the cover layer have been describedpreviously.

[0544] Referring back to FIG. 73, the conduction of a fluid through thechannel may be accomplished using a combination of hydrophobic andhydrophilic surfaces. In one embodiment, a series of hydrophobicsegments 1032 are applied to a surface of channels 1022 and 1042. Alayer of a hydrophilic material 1034 may be placed on the oppositesurface of the channel, with respect to the hydrophobic materials. Whenan aqueous fluid sample is introduced into the channel, the water isattracted toward the hydrophilic layer while being repelled by thehydrophobic layer. This attraction/repulsion creates a current withinthe channel. The hydrophilic surfaces may be composed of silicon orhexamethyldisilane. The hydrophobic surfaces may be composed of silicondioxide, silicon nitride, silicon dinitride, siloxane, or siliconoxynitride.

[0545] The system depicted in FIG. 73 may cause a current to flow in adirection from the left side toward the right. Thus the fluid,introduced at inlet 1040, may flow through the channel 1042 in adirection toward the particles. After contacting the particles, thefluid may pass thorough the cavity and into the lower channel 1022. Thehydrophilic and hydrophobic portions of the lower channel may induce acurrent that cause the fluid to flow toward the outlet of the sensorarray system.

[0546] Likewise, the system depicted in FIG. 77 may cause current toflow in a direction from the left side to toward the right.Alternatively, the fluid may exit through the top portion of the systemthrough the cover. The fluid may be introduced at inlet 1060 and mayflow through channel 1062. Fluid may then flow through cavity 1064 pastparticle 1065. The fluid may also flow through cavity 1066 past particle1067. The wall 1072 prevents the fluid from flowing past cavity 1066 inthe in channel 1062. After flowing through the cavities, the fluid flowsthrough channel 1068 and then up through cavity 1070. The hydrophilicand hydrophobic portions of the lower channel may induce a current thatcause the fluid to flow toward the outlet 1074 of the sensor arraysystem. In addition, FIG. 73 depicts a bubble-trap 1035 that may consistof a wall in a hydrophobic region.

[0547] The sensor array may be formed from a plurality of layers. Thelayers may be assembled with dry film materials and ultraviolet curableepoxy. The support layer serves as a base for the system. The supportlayer may be formed of a variety of materials including, but not limitedto glass, silicon nitride, silicon, silicon dioxide, plastic, and dryfilm photoresist. The support layer is depicted in FIG. 75D.

[0548] Onto the support layer is formed a spacer layer. The pattern foran embodiment of the spacer layer is depicted in FIG. 75C. The spacerlayer may be placed in the locations that will not be directly under thecavities. The spacer layer may allow a channel to be formed under thesensor array.

[0549] The sensor layer is formed upon the spacer layer. A pattern forthe etching of the sensor layer is depicted in FIG. 75B. The shadedareas 1031 represent the portion of the sensor layer that is etched to athickness that is less than the remaining portion of the sensor layer1033. The sensor layer may be formed from a variety of materials,including silicon, plastic, and dry film photoresist, as has beendescribed before. The sensor layer may be aligned with the support layerto allow a channel to be formed under the cavities. The channel mayallow fluids to pass from the sensor array system.

[0550] A cover layer is placed over the sensor layer. The etching of thecover layer may allow an upper channel 1042 to be formed between thesensor layer and the cover layer. The cover layer, in one embodiment,includes an opening 1052 that allows a fluid to be passed through thecover layer to the sensor layer. A pattern for the cover layer isdepicted in FIG. 75A. The opening may be aligned with a reservoirsection of the sensor layer.

[0551] In general, the use of a passive fluid transport system allowsonly a single use of the sensor array. Although the sensor array mayhave many chemical particles, and hence has multi-analyte capability,the surface wetting “pump” may only be used once. For many testingsituations (e.g., medical testing) this is not a significant problem,since it is desirable to dispose of the sensing element after a singleuse. If multiple testing of samples is to be performed an “array ofarrays” may be used, as depicted in FIG. 76. In this case, multiplesample introduction sites, each coupled to its own suite of sensorsites, may be fabricated. This setup may allow multiple uses of thesensor array (i.e., use one sensor suite for each test) or allow thesimultaneous analysis of multiple samples.

[0552] 4. Portable Sensor Array System

[0553] A sensor array system becomes most powerful when the associatedinstrumentation may be delivered and utilized at the application site.That is, rather than remotely collecting the samples and bringing themto a centrally based analysis site, it may be advantageous to be able toconduct the analysis at the testing location. Such a system may be use,for example, for point of care medicine, on site monitoring of processcontrol applications, military intelligence gathering devices,environmental monitoring, and food safety testing.

[0554] An embodiment of a portable sensor array system is depicted inFIG. 78. The portable sensor array system would, in one embodiment, havea size and weight that would allow the device to be easily carried by aperson to a testing site. The portable sensor array system includes alight source, a sensor array, and a detector. The sensor array, in someembodiments, is formed of a supporting member which is configured tohold a variety of particles in an ordered array. The particles are, insome embodiments, elements which will create a detectable signal in thepresence of an analyte. The particles may include a receptor moleculecoupled to a polymeric bead. The receptors may be chosen for interactingwith specific analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles.The supporting member may include a plurality of cavities. The cavitiesmay be formed such that at least one particle is substantially containedwithin the cavity. The sensor array has been previously described ingreater detail.

[0555] The portable sensor array system may be used for a variety ofdifferent testing. The flexibility of the sensor array system, withrespect to the types of testing, may be achieved through the use of asensor array cartridge. Turning to FIG. 78, a sensor array cartridge1010 may be inserted into the portable sensor array system 1000 prior totesting. The type of sensor array cartridge used will depend on the typeof testing to be performed. Each cartridge will include a sensor arraywhich includes a plurality of chemically sensitive particles, each ofthe particles including receptors specific for the desired task. Forexample, a sensor array cartridge for use in medical testing fordiabetes may include a number of particles that are sensitive to sugars.A sensor array for use in water testing, however, would includedifferent particles, for example, particles specific for pH and/or metalions.

[0556] The sensor array cartridge may be held in place in a manneranalogous to a floppy disk of a computer. The sensor array cartridge maybe inserted until it snaps into a holder disposed within the portablesensor system. The holder may inhibit the cartridge from falling outfrom the portable sensor system and place the sensor in an appropriateposition to receive the fluid samples. The holder may also align thesensor array cartridge with the light source and the detector. A releasemechanism may be incorporated into the holder that allows the cartridgeto be released and ejected from the holder. Alternatively, the portablesensor array system may incorporate a mechanical system forautomatically receiving and ejecting the cartridge in a manner analogousto a CD-ROM type system.

[0557] The analysis of simple analyte species like acids/bases, salts,metals, anions, hydrocarbon fuels, solvents may be repeated using highlyreversible receptors. Chemical testing of these species may berepeatedly accomplished with the same sensor array cartridge. In somecases, the cartridge may require a flush with a cleaning solution toremove the traces from a previous test. Thus, replacement of cartridgesfor environmental usage may be required on an occasional basis (e.g.,daily, weekly, or monthly) depending on the analyte and the frequency oftesting

[0558] Alternatively, the sensor array may include highly specificreceptors. Such receptors are particularly useful for medical testing,and testing for chemical and biological warfare agents. Once a positivesignal is recorded with these sensor arrays, the sensor array cartridgemay need to be replaced immediately. The use of a sensor array cartridgemakes this replacement easy.

[0559] Fluid samples may be introduced into the system at ports 1020 and1022 at the top of the unit. Two ports are shown, although more portsmay be present. One 1022 may be for the introduction of liquids found inthe environment and some bodily fluids (e.g., water, saliva, urine,etc.). The other port 1020 may be used for the delivery of human wholeblood samples. The delivery of blood may be accomplished by the use of apinprick to pierce the skin and a capillary tube to collect the bloodsample. The port may be configured to accept either capillary tubes orsyringes that include blood samples.

[0560] For the collection of environmental samples, a syringe 1030 maybe used to collect the samples and transfer the samples to the inputports. The portable sensor array system may include a holder that allowsthe syringe to be coupled to the side of the portable sensor arraysystem. One of the ports 1020 may include a standard luer lock adapter(either male or female) to allow samples collected by syringe to bedirectly introduced into the portable sensor array system from thesyringe.

[0561] The input ports may also be used to introduce samples in acontinuous manner. The introduction of samples in a continuous mannermay be used, e.g., to evaluate water streams. An external pump may beused to introduce samples into the portable sensor array system in acontinuous manner. Alternatively, internal pumps disposed within theportable sensor array system may be activated to pull a continuousstream of the fluid sample into the portable sensor array system. Theports are also configured to allow the introduction of gaseous samples.

[0562] In some cases it may be necessary to filter a sample prior to itsintroduction into the portable sensor array system. For example,environmental samples may be filtered to remove solid particles prior totheir introduction into the portable sensor array system. Commerciallyavailable nucleopore filters 1040 anchored at the top of the unit may beused for this purpose. In one embodiment, filters 1040 may have luerlock connections (either male or female) on both sides allowing them tobe connected directly to an input port and a syringe.

[0563] In one embodiment, all of the necessary fluids required for thechemical/biochemical analyses are contained within the portable sensorarray system. The fluids may be stored in one or more cartridges 1050.The cartridges 1050 may be removable from the portable sensor arraysystem. Thus, when a cartridge 1050 is emptied of fluid, the cartridgemay be replaced by a new cartridge or removed and refilled with fluid.The cartridges 1050 may also be removed and replaced with cartridgesfilled with different fluids when the sensor array cartridge is changed.Thus, the fluids may be customized for the specific tests being run.Fluid cartridges may be removable or may be formed as an integral partof the reader.

[0564] The fluid cartridges 1050 may include a variety of fluids for theanalysis of samples. In one embodiment, each cartridge may include up toabout 5 mL of fluid and be used for about 100 tests before beingdepleted. One or more of the cartridges 1050 may include a cleaningsolution. The cleaning solution may be used to wash and/or recharge thesensor array prior to a new test. In one embodiment, the cleaningsolution may be a buffer solution. Another cartridge 1050 may includevisualization agents. Visualization agents may be used to create adetectable signal from the particles of the sensor array after theparticles interact with the fluid sample. In one embodiment,visualization agents include dyes (visible or fluorescent) or moleculescoupled to a dye, which interact with the particles to create adetectable signal. In an embodiment, a cartridge 1050 may be a vacuumreservoir. The vacuum reservoir may be used to draw fluids into thesensor array cartridge. The vacuum cartridge would act in an analogousmanner to the vacutainer cartridges described previously. In anotherembodiment, a fluid cartridge may be used to collect fluid samples afterthey pass through the sensor array. The collected fluid samples may bedisposed of in an appropriate manner after the testing is completed.

[0565] In one embodiment, an alpha-numeric display screen 1014 may beused to provide information relevant to the chemistry/biochemistry ofthe environment or blood samples. Also included within the portablesensor array system is a data communication system. Such systems includedata communication equipment for the transfer of numerical data, videodata, and sound data. Transfer may be accomplished using either data oranalog standards. The data may be transmitted using any transmissionmedium such as electrical wire, infrared, RF and/or fiber optic. In oneembodiment, the data transfer system may include a wireless link (notshown) that may be used to transfer the digital chemistry/biochemistrydata to a closely positioned communications package. In anotherembodiment, the data transfer system may include a floppy disk drive forrecording the data and allowing the data to be transferred to a computersystem. In another embodiment, the data transfer system may include aserial or parallel port connection hardware to allow transfer of data toa computer system.

[0566] The portable sensor array system may also include a globalpositioning system (“GPS”). The GPS may be used to track the area that asample is collected from. After collecting sample data, the data may befed to a server, which compiles the data along with GPS information.Subsequent analysis of this information may be used to generate achemical/biochemical profile of an area. For example, tests of standingwater sources in a large area may be used to determine the environmentaldistribution of pesticides or industrial pollutants.

[0567] Other devices may also be included in the portable sensor arraythat are specific for other applications. For example, for medicalmonitoring devices including but not limited to EKG monitors, bloodpressure devices, pulse monitors, and temperature monitors.

[0568] The detection system may be implemented in a number of differentways such that all of the detection components fit within the casing ofthe portable sensor array system. For the optical detection/imagingdevice, either CMOS or CCD focal plane arrays may be used. The CMOSdetector offers some advantages in terms of lower cost and powerconsumption, while the CCD detector offers the highest possiblesensitivity. Depending on the illumination system (see below), eithermono-chrome or color detectors may be used. A one-to-one transfer lensmay be employed to project the image of the bead sensor array onto thefocal plane of the detector. All fluidic components may be sealed awayfrom contact with any optical or electronic components. Sealing thefluids away from the detectors avoids complications that may arise fromcontamination or corrosion in systems that require direct exposure ofelectronic components to the fluids under test. Other detectors such asphotodiodes, cameras, integrated detectors, photoelectric cells,interferometers, and photomultiplier tubes may be used.

[0569] The illumination system for colorimetric detection may beconstructed in several manners. When using a monochrome focal planearray, a multi-color, but “discrete-wavelength-in-time” illuminationsystem may be used. The simplest implementation may include severalLED's (light emitting diodes) each operating at a different wavelength.Red, green, yellow, and blue wavelength LEDs are now commerciallyavailable for this purpose. By switching from one LED to the next, andcollecting an image associated with each, colorimetric data may becollected.

[0570] It is also possible to use a color focal plane detector array. Acolor focal plane detector may allow the determination of calorimetricinformation after signal acquisition using image processing methods. Inthis case, a “white light” illuminator is used as the light source.“White light” LEDs may be used as the light source for a color focalplane detector. White light LEDs use a blue LED coated with a phosphorto produce a broad band optical source. The emission spectrum of suchdevices may be suitable for calorimetric data acquisition. A pluralityof LEDs may be used. Alternatively a single LED may be used.

[0571] Other light sources that may be useful include electroluminescentsources, fluorescent light sources, incandescent light sources, laserlights sources, laser diodes, arc lamps, and discharge lamps. The systemmay also be configured to use external light source (both natural andunnatural) for illumination.

[0572] A lens may be positioned in front of the light source to allowthe illumination area of the light source to be expanded. The lens mayalso allow the intensity of light reaching the sensor array to becontrolled. For example the illumination of the sensor array may be mademore uniform by the use of a lens. In one example, a single LED lightmay be used to illuminate the sensor array. Examples of lenses that maybe used in conjunction with an LED include Diffusing plate PN K43-717Lens JML, PN61874 from Edmund scientific.

[0573] In addition to calorimetric signaling, chemical sensitizers maybe used that produce a fluorescent response. The detection system maystill be either monochrome (for the case where the specific fluorescencespectrum is not of interest, just the presence of a fluorescence signal)or color-based (that would allow analysis of the actual fluorescencespectrum). An appropriate excitation notch filter (in one embodiment, along wavelength pass filter) may be placed in front of the detectorarray. The use of a fluorescent detection system may require anultraviolet light source. Short wavelength LEDs (blue to near UV), maybe used as the illumination system for a fluorescent based detectionsystem.

[0574] In some embodiments, use of a light source may not be necessary.The particles may rely on the use of chemiluminescence,thermoluminescence or piezoluminescence to provide a signal. In thepresence of an analyte of interest, the particle may be activated suchthat the paticles produce light. In the absence of an analyte, theparticles may not exhibit produce minimal or no light.

[0575] The portable sensor array system may also include an electroniccontroller which controls the operation of the portable sensor arraysystem. The electronic controller may also be capable of analyzing thedata and determining the identity of the analytes present in a sample.While the electronic controller is described herein for use with theportable sensor array system, it should be understood that theelectronic controller may be used with an of the previously describedembodiments of an analyte detection system.

[0576] The controller may be configured to control the variousoperations of the portable sensor array. Some of the operations that maybe controlled or measured by the controller include: (i) determining thetype of sensor array present in the portable sensor array system; (ii)determining the type of light required for the analysis based on thesensor array; (iii) determining the type of fluids required for theanalysis, based on the sensor array present; (iv) collecting the dataproduced during the analysis of the fluid sample; (v) analyzing the dataproduced during the analysis of the fluid sample; (vi) producing a listof the components present in the inputted fluid sample (vii) monitoringsampling conditions (e.g., temperature, time, density of fluid,turbidity analysis, lipemia, bilirubinemia, etc).

[0577] Additionally, the controller may provide system diagnostics andinformation to the operator of the apparatus. The controller may notifythe user when routine maintenance is due or when a system error isdetected. The controller may also manage an interlock system for safetyand energy conservation purposes. For example, the controller mayprevent the lamps from operating when the sensor array cartridge is notpresent.

[0578] The controller may also be configured to interact with theoperator. The controller preferably includes an input device 1012 and adisplay screen 1014. A number of operations controlled by thecontroller, as described above, may be dependent on the input of theoperator. The controller may prepare a sequence of instructions based onthe type of analysis to be performed. The controller may send messagesto the output screen to let the used know when to introduce samples forthe test and when the analysis is complete. The controller may displaythe results of any analysis performed on the collected data on theoutput screen.

[0579] Many of the testing parameters may be dependent upon the type ofsensor array used and the type of sample being collected. The controllerwill, in some embodiments, require the identity of the sensor array andtest being performed in order to set up the appropriate analysisconditions. Information concerning the sample and the sensor array maybe collected in a number of manners. In one embodiment, the sample andsensor array data may be directly inputted by the user to thecontroller. Alternatively, the portable sensor array may include areading device which determines the type of sensor cartridge being usedonce the cartridge is inserted. In one embodiment, the reading devicemay be a bar code reader which is configured to read a bar code placedon the sensor array. In this manner the controller can determine theidentity of the sensor array without any input from the user. In anotherembodiment, the reading device may be mechanical in nature. Protrusionsor indentation formed on the surface of the sensor array cartridge mayact as a code for a mechanical reading device. The information collectedby the mechanical reading device may be used to identify the sensorarray cartridge. Other devices may be used to accomplish the samefunction as the bar code reader. These devices include, but are notlimited to, smartcard readers and RFID systems.

[0580] The controller may also accept information from the userregarding the type of test being performed. The controller may comparethe type of test being performed with the type of sensor array presentin the portable sensor array system. If an inappropriate sensor arraycartridge is present, an error message may be displayed and the portablesensor array system may be disabled until the proper cartridge isinserted. In this manner, incorrect testing resulting from the use ofthe wrong sensor cartridge may be avoided.

[0581] The controller may also monitor the sensor array cartridge anddetermine if the sensor array cartridge is functioning properly. Thecontroller may run a quick analysis of the sensor array to determine ifthe sensor array has been used and if any analytes are still present onthe sensor array. If analytes are detected, the controller may initiatea cleaning sequence, where a cleaning solution is passed over the sensorarray until no more analytes are detected. Alternatively, the controllermay signal the user to replace the cartridge before testing isinitiated.

[0582] Another embodiment of a portable sensor array system is depictedin FIGS. 79A and 79B. The portable sensor array 1100 includes a body1110 that holds the various components used with the sensor arraysystem. A sensor array, such as the sensor arrays described herein, maybe placed in cartridge 1120. Cartridge 1120 may support the sensor arrayand allow the proper positioning if the sensor array within the portablesensor system.

[0583] A schematic cross-sectional view of the body of the portablesensor array system is depicted in FIG. 79B. The cartridge 1120, inwhich the sensor array is disposed, extends into the body 1110. Withinthe body, a light source 1130 and a detector 1140 are positionedproximate to the cartridge 1120. When the cartridge 1120 is insertedinto the reader, the cartridge may be held, by the body 110, at aposition proximate to the location of the sensor array within thecartridge. The light source 1130 and detector 1140 may be used analyzesamples disposed within the cartridge. An electronic controller 1150 maybe coupled to detector. The electronic controller 1150 may be configuredto receive data collected by the portable sensor array system. Theelectronic controller may also be used to transmit data collected to acomputer.

[0584] An embodiment of a cartridge for use in a sensor array system isdepicted in FIG. 80. The cartridge include a carrier body 1210,.that isformed of a material that is substantially transparent to a wavelengthof light used by the detector. IN one embodiment, plastic materials maybe used. Examples of plastic materials that may be used includepolycarbonates and polyacrylates. In one embodiment the body may beformed from Cyrolon AR2 Abrasion Resistant polycarbonate sheet atthicknesses of 0.118 inches and 0.236 inches. A sensor array gasket 1220may be placed on the carrier body 120. The sensor array gasket 1220, mayhelp reduce or inhibit the amount of fluids leaking from the sensorarray. Leaking fluids may interfere with the testing being performed.

[0585] A sensor array 1230 may be placed onto the sensor array gasket.The sensor array may include one or more cavities, each of whichincludes one or more particles disposed within the cavities. Theparticles may react with an analyte present in a fluid to produce adetectable signal. Any of the sensor arrays described herein may be usedin conjunction with the portable reader.

[0586] A second gasket 1240, may be positioned on the sensor array. Thesecond gasket 1240, may be disposed between the sensor array 1230 and awindow 1250. The second gasket 1240 may form a seal inhibiting leakageof the fluid from the sensor array. The window may be disposed above thegasket to inhibit damage to the sensor array.

[0587] The assembly may be completed by coupling a cover 1270 to thebody 1210. A rubber spring 1260 may be disposed between the cover andthe window to reduce pressure exerted by the cover on the window. Thecover may seal the sensor array, gaskets, and window into the cartridge.The sensor array, gaskets and window may all be sealed together using apressure sensitive adhesive. Examples of a pressure sensitive adhesiveinclude Optimount 237 made by Seal products. Gaskets may be made frompolymeric materials. In one example, Calon II—High Performance materialfrom Arlon may be used. The rubber spring may be made form a siliconrubber material.

[0588] The cover may be removable or sealed. When a removable cover isused the cartridge may be reused by removing the cover and replacing thesensor array. Alternatively, the cartridge may be a one use cartridge inwhich the sensor array is sealed within the cartridge.

[0589] The cartridge may also include a reservoir 1270. The reservoirmay be configured to hold the analyte containing fluid after the fluidspass through the sensor array. FIG. 81 depicts a cut away view of thecartridge that shows the positions of channels formed in the cartridge.The channels may allow the fluids to be introduced into the cartridge.The channels also may conduct the fluids from the inlet to the sensorarray and to the reservoir.

[0590] In one embodiment, the cartridge body 1210, includes a number ofchannels disposed throughout the body. An inlet port 1282 is configuredto receive a fluid delivery device for the introduction of fluid samplesinto the cartridge. In one embodiment, the inlet port may include a luerlock adapter, configured to couple with a corresponding luer lockadapter on the fluid delivery device. For example, a syringe may be usedas the fluid delivery device. The luer lock fitting on the syringe maybe coupled with a mating luer lock fitting on the inlet port 1282. Luerlock adapters may also be coupled to tubing, so that fluid delivery maybe accomplished by the introduction of fluids through appropriate tubingto the cartridge.

[0591] The introduced fluid passes through channel 1284 to channeloutlet 1285. Channel outlet 1285 may be coupled to an inlet port on asensor array (see description of sensor arrays herein). Channel outlet1285 is also depicted on FIG. 80. The fluids travels into the sensorarray and through the cavities. After passing through the cavities, thefluid exits the sensor array and enters channel 1286 via channel inlet1287. The fluid passes through channel 1286 to reservoir 1280. Tofacilitate the transfer of fluids through the cartridge, the reservoirmay include an air outlet port 1288. Air outlet port 1288 may beconfigured to allow air to pass out of the reservoir, while retainingany fluids disposed within the reservoir. In one embodiment, the airoutlet port 1288 may be an opening formed in the reservoir that iscovered by a semipermeable membrane. A commercially available air outletport includes a DURAVENT container vent, available from W. L. Gore. Itshould be understood, however, that any other material that allows airto pass out of the reservoir, while retaining fluids in the reservoirmay be used. After extended use the reservoir 1280 may become filledwith fluids. An outlet channel 1290 may also be formed extending throughthe body 1210 to allow removal of fluids from the body. Fluid cartridges1292 for introducing additional fluids into the sensor array may beincorporated into the cartridges.

[0592] Magnetic Particle Production and Use

[0593] Magnetic particles may be made by different methods. In anembodiment, a solution containing Fe(II) and Fe(III) (typically FeCl₂and FeCl₃), and a polymer (e.g. a protein) having available coordinationsites may be treated (by titration or otherwise) with a strong base suchas aqueous ammonia in order to precipitate magnetic iron oxides such asmagnetite (Fe₃O₄) in a form which is intimately combined with thepolymer. The precipitation may be typically carried out with rapidstirring and optional agitation by sonication, in order to produceresuspendable magnetic-polymer particles.

[0594] After precipitation, the particles may be washed and subsequentlyresuspended in a buffer solution at approximately neutral pH. Otherembodiments may involve the use of metals other than iron in thecoprecipitation reaction. In particular, Fe(III) may be replaced by anyof a wide range of transition metal ions. In some cases, iron may becompletely supplanted by appropriately selected transition metal ions.In some cases, the use of metals other than iron produces coloredparticles ranging from white to dark brown.

[0595] Magnetic-polymer particles may be produced of varying size.Magnetic particles may be tailor-made to include specific biofunctionalligands useful in various analytical, diagnostic, and otherbiological/medical applications. Magnetic particles may be produced withselect chemical reagents that may be useful in various analyticalapplications.

[0596] Subsequent to precipitation and resuspension of themagnetic-polymer particles, they may be treated with a bifunctionalreagent in order to cross-link reactive sites present on the polymer.This cross-linking may be effective as either an intra-particulatecross-linking in which reactive sites are bound on the same particle, ormay be a reaction of an extra-particulate ligand which may then becross-linked to the polymer on a given particle. In the second case, abifunctional reagent having a relatively short distance between its twofunctional groupings may be desirable to promote linkage between theparticle polymer and the extra-particulate species. Conversely,intra-particulate cross-linking may be promoted by the use of abifunctional reagent which may be longer and may not be stericallyhindered from bending so that two reactive sites on a single particlemay be linked by a single bifunctional molecule.

[0597] As an alternative to the use of sonication during either theprecipitation or resuspension steps outlined above, another type ofagitation (such as mechanical stirring) may be employed.

[0598] Resuspension of the magnetic-polymer particles may be typicallycarried out in a low ionic strength buffer system (e.g. 40 mMphosphate). The buffer system may enable resuspension of particles whichare not resuspendable in non-ionic solutions. In addition to phosphatebuffers, borate and sulfate systems may also be used. The association ofpolymer and metal may result from coordination of metal present duringcoprecipitation by coordination sites on the polymer. It may be thatcertain coordination sites are more “available” than others, based onboth the strength of the coordinate bond which may be formed by theparticular atom, and the spatial hindrances imposed by surroundingatoms. It is known, for instance, that oxygen atoms having a “free”electron pair complex iron more strongly than amine nitrogen atoms and,to an even greater degree, a hydroxyl oxygen atom. Thus, a polymerbearing oxy-acid functional groups may provide better product particlesthan an amine-substituted polymer. Similarly, coordination sites whichmay be freely approached to close distances may yield better performancethan sites which are hindered in either a path of approach or inapproach distance.

[0599] The above-described trends may be qualitatively observable invarious experiments. The presence of “available coordination sites”appears necessary to the production of the resuspendablemagnetic-polymer particles. For example, such diverse polymers asnatural proteins, synthetic proteins, poly-amino acids,carboxy-poly-alkyls, alkoxy-poly-alkyls, amino-poly-alkyls,hydroxy-poly-alkyls, and various copolymers of these have all beendemonstrated to produce suitable particles. In addition, other polymerssuch as sulfoxy-poly-alkyls, poly-acrylamines, poly-acrylic acid, andsubstituted poly-alkylenes may produce similar particles.

[0600] In selecting the transition metals to be employed in thecoprecipitation reaction, several criteria may be important. First, thefinal compound must have one or more unpaired electrons in itsstructure. Second, one of the metals must possess an availablecoordination site for bonding to a polymer. Third, the coprecipitatemust be capable of forming a cubic close-packed or hexagonalclose-packed (eg. for cubic: spinel or inverse spinel) crystallinestructure. This last requirement may be due to the need for a very closepacking in order for a compound to be magnetic.

[0601] In an embodiment, polymers useful in preparing the magneticparticles may be “tailor-made” to include monomers which may exhibit aspecific biofunctional activity. Using such a polymer may permit directprecipitation of a biofunctional magnetic-polymer particle which mayrequire little or no further treatment in order to be useful in assayswhich rely on the particular biofunctional activity of the polymer.

[0602] In some embodiments, larger, less stable particles may be useful.The particles may be made to agglomerate while still retaining boththeir biofunctional and magnetic characteristics. Agglomeration of theparticles may be accomplished by treatment of a suspension with apredetermined amount of, for example, barium chloride solution. Thistreatment may be designed to cause the particles to settle out ofsuspension in a predetermined period of time in order to allow theperformance of further procedures, or to allow the larger particles tobe easily attracted by relatively small magnets. U.S. Pat. No. 4,795,698to Owen et al., which is incorporated herein by reference, providesfurther details for producing magnetic particles.

[0603] Magnetic particles may also be produced from metallocenes andmetal hydroxide compounds. These particles may then be incorporated intopolymeric materials to produce magnetically active particles.

[0604] Metallocenes are cycopentadienyl coordinate complexes of metals.The cyclopentadienyl group, C₅H₅, has long been known to form complexeswith metals or metalloidal atoms. In an embodiment, metallocenes may becyclopentadienyl complexes of transition metals. The transition-metalsmay include, for example, iron (Fe), magnesium (Mg), manganese (Mn),cobalt (Co), nickel (Ni), zinc (Zn) and copper (Cu). Particularly usefulmetallocenes may be ferrocene (C₅H₅)₂Fe, nickelocene, (C₅H₅)₂Ni, andcobaltocene, (C₅H₅)₂Co. Metalloc formula (C₅H₅)₂ M, wherein M is themetal and have a “sandwich” configuration. The structure of metallocenesendows these molecules with high thermal stability (e.g., up to about500° C. for ferrocene).

[0605] In an embodiment, an aqueous slurry of the metallocene may beproduced. The slurry may be prepared, for example, by combining themetallocene compound and water, and mixing or by milling in a highenergy mill, such as a sand mill or a ball mill. The length of time forwhich the slurries are milled will depend upon the particle size of theproduct which may be desired. The slurry may generally contain fromabout 0.1 to about 40 percent (%) by weight of the metallocene. A slurrycontaining from about 20 to about 25% by weight metallocene may beparticularly useful.

[0606] The aqueous metallocene slurry may be combined with a secondaqueous slurry of a metal hydroxide. The choice of metal hydroxide maydepend upon the properties of the particles which may be desired. Forexample, to produce magnetite particles, iron (II) hydroxide (ferroushydroxide) may be used. Other metal hydroxides which may be used toproduce magnetic particles may include cobalt (II) hydroxide, cobalt(III) hydroxide, iron (III) hydroxide and nickel hydroxide. Slurries ofthese metal hydroxides may be prepared by precipitating a salt of themetal (e.g. chloride or sulfate salt) in an aqueous medium using a base,such as sodium hydroxide or ammonium hydroxide. An aqueous iron (II)hydroxide slurry may be prepared by precipitating an aqueous solution offerrous chloride or ferrous sulfate with ammonium or sodium hydroxide toform ferrous hydroxide (FeO(OH)). The resulting gelatinous precipitateof iron (II) hydroxide may be filtered, and the solid material may becollected, combined with water and milled in a high energy mill to formthe slurry. The metal hydroxide slurry may contain from about 0.1 toabout 40 percent (%) by weight of the metal hydroxide.

[0607] The two slurries may be combined and the mixture may be milled ina high energy mill, such as a commercial ball or sand mill, for a periodof time sufficient to form fine magnetic particles, generally for about1 hour to about 60 hours. Generally, the longer the milling step, thesmaller the particles which may be formed.

[0608] In an embodiment, magnetite particles may be formed from iron(II) hydroxide and ferrocene according to the following equation:

2FeO(OH)+Fe(C₅H₅)₂→Fe₃O₄+2(C₅H₅)+H₂O+H₂ (gas)

[0609] The iron (II) hydroxide powder may be milled in intimate contactwith the ferrocene. Over a period of about 20 to 40 hours, the twomaterials may react by slow dissociation of the hydroxide to formmagnetite, free cyclopentene, water and hydrogen. It may be necessary toallow sufficient void space in the mill, or to vent the millperiodically to accommodate the release of the hydrogen gas formedduring the reaction. The particles may then be isolated and incorporatedinto polymeric materials to produce beads comprising magnetic particles.Additional production details may be found in U.S. Pat. No. 5,071,076 toChagnon et al., which is incorporated herein by reference.

[0610] In an embodiment, colloidal polymer or protein magnetite may beprepared with highly controllable, polymer/protein magnetite ratios.Typically, the particles may be precipitated from solutions of hydratedferric and ferrous chlorides at 3.5 and 1.5 mg/ml, respectively, withprotein content ranging from 500 ug/ml to 1.5 mg/ml. After appropriatewashing, resuspension and sonication of such precipitates, colloidal,magnetically responsive particles may be produced, wherein the meandiameter of particles may be approximately inversely proportional tostarting protein concentrations. Particles about 20 nanometers or lessin diameter may be obtained at the higher protein concentrations,whereas particles approximately 100 nanometers in diameter may beobtained at the lower end of the range of protein concentrations. It hasbeen found that the ease with which various of these colloidal solutionsmay be salted out may be inversely related to the protein concentrationof the solution and may be directly related to particle size. In otherwords, the smaller, higher protein containing particles may be moredifficult to salt out. These results suggest that the particles havinghigher protein concentration may be more lyophilic, which might beexpected because of the greater interaction between solvent water andprotein, as compared with magnetite. Other possible explanations forthis observed phenomenon may be that the magnetic cores of the largercolloidal particles may be easier to flocculate because of theirmagnetic moments, or that the smaller particles offer relatively largersurface area and consequently more surface charge to be neutralized.

[0611] In an embodiment, colloidal, magnetically responsive particlesbearing (i) a biospecific binding material having binding affinity forthe target substance of interest or (ii) a suitable retrieval agent, forexample, anti-fluorescein, where a fluoresceinated receptor for thetarget substance may be used, may be incubated with an appropriatelylabeled specific binding substance and test sample suspected ofcontaining the target substance, under conditions such thatagglomeration of such particles may not occur. Agglomeration may notoccur, for instance, because the binding capacity of the specificbinding substance or the concentration of the target substance in thetest medium may be too low. Following the binding of sufficient labeledsubstance (or inhibition thereof), an agglomerating agent, which may beeither non-specific, or specific, preferably the former, e.g., a simplesalt solution, may be added to the incubation mixture to causeagglomeration. Agglomeration may be brought about by the addition of asecond non-specific agglomerating agent, e.g., an appropriately chosencolloid, if desired.

[0612] Alternatively, agglomeration may be effected by means of aspecific agglomerating agent capable of cross-linking a component of thecolloidal magnetic particles, such as a specific antibody. The resultingagglomerate may be removed from solution via centrifugation, filtrationor, via magnetic separation. It may also be possible to use anon-specific and/or specific agglomerating agents in variouscombinations, if desired. Thus, second colloid addition plus salting outmay be feasible, as may the use of a second magnetically responsivecolloidal particle bearing a receptor capable of cross-linking with asubstance present on the colloidal protein magnetite initially added tothe test sample.

[0613] Another useful application of the conversion of colloidalmaterial to a magnetically separable form by the addition of a secondcolloid, may be to use protein colloidal magnetite as the agglomeratingagent for some other non-magnetic colloidal material, where the latterbears the target substance of interest.

[0614] Colloidal reagents and non-specific or specific agglomeratingagents may be added to the test medium simultaneously, rather thansequentially, as previously described. This may be accomplished byadding a suitable agglomerating agent to one of the colloidal reagentsused in the assay, so that conversion of the colloid takes place after asubstantial level of ligand/receptor interaction has occurred. Furtherinformation on production of magnetic colloidal particles may be foundin U.S. Pat. No. 5,108,933 to Liberti et al., which is incorporatedherein by reference.

[0615] In an embodiment, permanently magnetized materials may be used toproduce magnetic particles. Previously discussed agglomerationtechniques may be used to form particles in which the particlecomposition may encapsulate the magnetic material. In an embodiment, themagnetic material may be suspended in a solution from which theparticles may be formed. As the particles begin to form, due toagglomeration or other methods, the suspended magnetic material may beencapsulated thereby forming a magnetic particle. Magnetic material mayalso be incorporated into particles by physical means. In an embodiment,magnetic materials may be intermixed with particles using methods suchas, but not limited to ball mills, low intensity mixers, and pug mills.A wide variety of magnetized materials may be used in the magneticparticles. Examples of magnetized materials, besides those materialspreviously discussed, may include, but are not limited to materials suchas alnico, ferrite, barium ferrite, strontium ferrite, neodymium ironboron, samarium cobalt, iron oxide, or other ferromagnetic materials.

[0616] Upon formation of the magnetic particle, the magnetic particlemay be further modified with target analyte materials. Eventually, themagnetic particles may be placed within the sensor array. In anembodiment, the magnetic particles may be located within the cavity orcavities of a sensor array by placement of permanent magnets in such amanner that the magnetic particle may be directed to a particularlocation, in this instance, a cavity in the sensor array. In anembodiment, a permanent magnet may be located under a cavity ofinterest. A solution containing suspended magnetic particles may beallowed to flow over the cavity, wherein a magnetic particle may bedirected into the cavity by the interaction of the magnetic particle andthe permanent magnet. Depending upon the cavity size, other particlesmay or may not be directed into the cavity. For example, a cavity onlylarge enough to include one magnetic particle, may capture one particle,but, based upon space limitation, no further particles may be directedinto the cavity. Conversely, a cavity large enough to include severalparticles may have several particles directed toward it before thecavity may no longer capture particles. When the desired cavity orcavities may be filled, a cover layer may be added to the substrate toretain the particles as discussed in previous sections. Directingmagnetic particles to magnets for collection or to a particular locationare further discussed in U.S. Pat. No. 4,813,277 to Miller, et al, whichis incorporated herein by reference.

[0617] Permanent magnets may be used to direct magnetic particles intocavities, but other embodiments may be possible. In an embodiment,electromagnets may be located at a desired cavity, such that themagnetic particle may be drawn into the desired cavity. For example, aflow of magnetic particles may be allowed to pass over the sensor array.An electromagnet may be located under a cavity such that as energy maybe supplied to the electromagnet, a flowing magnetic particle may bedirected into the desired cavity. A plurality of cavities may be locatedon the sensor array and a discrete electromagnet may be assigned to eachcavity. Current flow to each electromagnet may be monitored such that amagnetic particle or particles may be directed to individual cavities.By controlling the electrical current to the electromagnets, somecavities may be filled with magnetic particles while other cavities mayremain empty. A second flow of different magnetic particles may beallowed to flow over the sensor array, at which time otherelectromagnets may be activated thereby causing the different magneticparticles to be directed into the currently empty cavities. Thisprocedure may continue using other different magnetic particles untilthe selected cavities may be filled. In this way, various cavities maybe filled with different magnetic particles. Other embodiments may allowlocation of multiple magnetic particles within the same cavity therebyproviding the ability to detect multiple analytes from the same cavity.Other variations of cavities and particles may be possible wherein thevariations may not be limited by the foregoing embodiments. U.S. Pat.No. 5,981,297 to Baselt, which is incorporated herein by reference,further describes the recognition of magnetic particles with magnets

[0618] Formation of Cavities with Retaining Projections

[0619] In an embodiment, a mask may be deposited on a bulk crystalline(100) silicon substrate to form an integrated cover layer. The coverlayer may be, but is not limited to, silicon nitride, a plastic, silicondioxide, or a dry film photoresist material. The cover layer may beformed or etched in such a way that, after etching of the siliconsubstrate, various flexible micromachined projections may be present inthe cover layer. Many types of structures formed on the cover layer mayprovide for development of flexible projections after etching. Examplesof structures that may be formed on the cover layer may be, but are notlimited to: star; cross; circle; square; or any other type of formationthat provides for flexible projections after etching. In an embodiment,a cross, formed by an equal length upright with a transverse beam may beformed in the cover layer. An amount of the cover layer may be removedsuch that the substrate may be exposed to an etchant material. Afterremoving the desired amount of cover layer, the substrate may be etchedanisotropically. The etchant may continue to remove the siliconsubstrate until the bounding (111) planes may be reached. The resultingcavity may be a pyramidal shape into the silicon substrate. Thepyramidal shaped cavity may enhance fluid flow. The cavity may be formedsuch that a bottom opening may also be present.

[0620] The flexible projections formed from the undercutting of thesilicon substrate beneath the cover layer may provide a method ofretention of the particle. In an embodiment, the flexible projectionsmay be produced from a mask opening which may be smaller than theunderlying cavity. The particle may then be manipulated past theflexible projections into the cavity. As the particle passes theflexible projections, the flexible projections may be displaced downwarduntil the particle passes completely by the flexible projections andinto the cavity. As the particle passes the flexible projections, theflexible projections may return to their original position, therebyproviding retention of the particle in the cavity. Retention of theparticle in the cavity may be maintained by the flexible projectionsduring subsequent handling of the sensor array.

[0621]FIG. 82 shows the placement of a particle (1) into a cavity. Theparticle may be placed proximate to the cavity on top of the flexibleprojections (2) as shown in (a), at which time a micromanipulator may beused to press the particle past the flexible projections. The flexibleprojections may bend as the particle may be pressed past theprojections, as shown in (b) and (c), until the particle may be placedwithin the cavity. The flexible projections may return to their normalposition, as shown in (d), as the particle moves past the flexibleprojections and is substantially retained within the cavity.

[0622] The flexible projections may provide for specific size selectionof particles to be placed into the cavity. In an embodiment, it may beassumed the particles may have a gaussian distribution. In anon-limiting example, an opening may be provided in the cover layer bythe flexible projections which may be an opening larger than the meansize of the particle times a sigma value. The sigma value as definedhereinafter is the variability in size of a particle around a meanparticle diameter of a gaussian distribution of particles. The bottomopening of the cavity may be an opening smaller than the mean size ofthe particle times a sigma value. If a 10% sigma value of the particlediameters may be assumed and a 10% sigma value of the top and bottomcavity openings, only the next size up or down may have a chance offilling the cavity. Assuming these variables, the probability forplacing a particle the next size up in the cavity may be about one partin one thousand. The probability of placing a particle the next sizedown in the cavity may be about 1 in 300. Reduction in the variabilityof the particle size and reduction in the variability in the top andbottom openings of the cavity may result in a higher percentage ofcorrectly sized particles being placed in the cavity.

[0623] In an embodiment, another strategy which may be employed withbead capture selectivity probability may be the use of three cavities ofa desired size to provide triple redundancy. In this strategy, threecavities of the selected size may be used and selection criteriadesigned such that if two cavities contain the correct particle size,the cavities may be considered correctly filled. An error may result iftwo-same sized cavities may be incorrectly filled. The foregoingcriteria may provide for a selection of the probability of placing a toolarge particle of about 1 in 10⁶ and placement of a too small particleof about 1 in 77,000. Error rates may be further reduced by decreasingthe variability of the particle diameter and variability in the cavitytop and bottom openings.

[0624] In an embodiment, a particle may be placed in a cavity usingvarious techniques such as individual placement of particles.Micromanipulators may be used in the individual placement of a particle.A vacuum or flow system may be used to provide for more rapid productionof cavity arrays compared to individual placement of particles intocavities. In an embodiment, a wafer may be fabricated with the correcttop and bottom cavity openings to select the desired particle size. Asolution of particles with a wide range of size distribution may beproduced. The wafer may then be dipped into the solution whereupon avacuum or flow may pull the particles past the cavity top flexibleprojections. A too large particle may not pass the top opening and a toosmall particle may pass through the cavity and out the cavity bottom. Acorrectly sized particle may pass the top opening flexible projectionsand be retained on the cavity bottom. In the embodiment, the flexibleprojections may be used as cavity opening discriminators. Flexing of theprojections as the particle passes may not be necessary.

[0625] A combination of correctly sized flexible projections andparticles may be used to produce a backflow preventer and pump. In anembodiment, a cavity may be fabricated such that the slits in the coverlayer produce a rectangular bottom opening. The top layer may befabricated such that a round opening slightly smaller than the particlemay be produced. The flexible projections of the bottom opening may bedesigned for placement of a particle into the cavity. The fluid flow maybe inhibited or stopped if the flow direction forces the particleagainst the round top opening. If the flow is reversed, the particle maybe forced against the flexible projections and depending upon the designof the flexible projections, flow may occur or may be significantlyinhibited. For example, the flexible projections may be designed suchthat the slits may be as small as possible resulting in a significantdecrease in back-flow capabilities. The effect of this embodiment mayproduce a valve with a high flow coefficient for flow in one directionand a low flow coefficient in the opposite direction.

[0626] The flexible projections may be designed to bend in one directionmore favorably than in the opposite direction. In an embodiment,multiple lithography or deposition steps for producing the cover layermay provide a flexible projection which may flex preferably in thedirection to allow placement of a particle within the cavity. Theflexibility may be reduced in the direction in which the projections maybe required to flex for removal of the particle. Providing enhancedflexibility in only one flexural direction may allow reduction of slitsize in the cover layer needed to provide etch access to the siliconsubstrate.

[0627] In an embodiment, the flexible projections may be produced byundercutting the silicon substrate as described previously. The topcover flexible projections and bottom cover opening may be fabricated tothe diameter desired, such that a particle may only be accepted in ashrunken state. The particle to be placed within the cavity may beexposed to a medium in which the particle may be caused to shrink. Theshrunken particle may then be placed within the cavity at which pointthe particle may be exposed to a medium which causes the particle toreturn to it's normal diameter state. The particle may then be capturedwithin the cavity. Correctly designing the swollen state of the particleand the flexible projections, the particle may be retained within thecavity subsequent to further processing of the array.

[0628] The sensor array may be used as a method for sorting varioussized particles. In an embodiment, the sensor array may be fabricatedwith various sized cavities which may capture various sized particles.Depending upon etch time, the cavity sizes may be configured todifferent sizes. A shorter etch time may produce a smaller cavity sizebased upon the depth of the cavity into the substrate.

[0629] In an embodiment to provide selection of only one particle sizefrom a distribution of particle sizes, a solution of particles with awide range of particle size distribution may be allowed to flow over thesubstrate. A vacuum or flow may be used to pull the particles past thecavities etched into the substrate support. Those particles with a toolarge diameter may not be captured by a cavity where the top opening maybe smaller than the particle diameter. The too large particle maycontinue to flow across the sensor array. Those particles with a smallerdiameter than the bottom opening may be drawn into the cavity as theypass the top opening, but the small diameter particle may pass throughthe bottom opening and out of the substrate support. Particle sizessmaller than the top opening, but larger than the bottom opening may bedrawn into the cavity and retained within the cavity. Those particleslarger than the top opening and smaller than the bottom opening may notbe retained on the substrate support. The non-retained particles mayflow away from the substrate support. The flow may then be stopped andthe substrate along with the captured particles may be removed from theflow. A reverse flow may then be used to dislodge the particles into adesired location.

[0630] In an embodiment, the array may provide an ability to pick andplace many particles at once. The substrate may be fabricated with topand bottom openings designed to select a certain desired particle size.A solution of particles may be flowed over the substrate. Thoseparticles of the desired particle size may be captured by the cavitiesas discussed in the previous section. The flow may then be stopped andthe substrate, along with the captured particles, may be removed fromthe flow. A reverse flow may then be used to dislodge the particles intoa desired location.

[0631] Further modifications and alternative embodiments of variousaspects of the invention will be apparent to those skilled in the art inview of this description. Accordingly, this description is to beconstrued as illustrative only and is for the purpose of teaching thoseskilled in the art the general manner of carrying out the invention. Itis to be understood that the forms of the invention shown and describedherein are to be taken as the presently preferred embodiments. Elementsand materials may be substituted for those illustrated and describedherein, parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

What is claimed is:
 1. A system for detecting an analyte in a fluidcomprising: a light source; a sensor array, the sensor array comprisinga supporting member comprising at least one cavity formed within thesupporting member; a particle, the particle positioned within thecavity, wherein the particle is configured to produce a signal when theparticle interacts with the analyte during use; and a detector, thedetector being configured to detect the signal produced by theinteraction of the analyte with the particle during use; wherein thelight source and detector are positioned such that light passes from thelight source, to the particle, and onto the detector during use.
 2. Thesystem of claim 1, wherein the system comprises a plurality of particlespositioned within a plurality of cavities, and wherein the system isconfigured to substantially simultaneously detect a plurality ofanalytes in the fluid.
 3. The system of claim 1, wherein the systemcomprises a plurality of particles positioned within the cavity.
 4. Thesystem of claim 1, wherein the light source comprises a light emittingdiode.
 5. The system of claim 1, wherein the light source comprises awhite light source.
 6. The system of claim 1, wherein the sensor arrayfurther comprises a bottom layer and a top cover layer, wherein thebottom layer is coupled to a bottom surface of the supporting member,and wherein the top cover layer is coupled to a top surface of thesupporting member; and wherein both the bottom layer and the top coverlayer are coupled to the supporting member such that the particle issubstantially contained within the cavity by bottom layer and the topcover layer.
 7. The system of claim 1, wherein the sensor array furthercomprises a bottom layer and a top cover layer, wherein the bottom layeris coupled to a bottom surface of the supporting member, and wherein thetop cover layer is coupled to a top surface of the supporting member;and wherein both the bottom layer and the top cover layer are coupled tothe supporting member such that the particle is substantially containedwithin the cavity by bottom layer and the top cover layer, and whereinthe bottom layer and the top cover layer are substantially transparentto light produced by the light source.
 8. The system of claim 1, whereinthe sensor array further comprises a bottom layer coupled to thesupporting member, and wherein the supporting member comprises silicon,and wherein the bottom layer comprises silicon nitride.
 9. The system ofclaim 1, wherein the sensor array further comprises a sensing cavityformed on a bottom surface of the sensor array.
 10. The system of claim1, wherein the supporting member is formed from a plastic material, andwherein the sensor array further comprises a top cover layer, the topcover layer being coupled to the supporting member such that theparticle is substantially contained within the cavity, and wherein thetop cover layer is configured to allow the fluid to pass through the topcover layer to the particle, and wherein both the supporting member andthe top cover layer are substantially transparent to light produced bythe light source.
 11. The system of claim 1, further comprising a fluiddelivery system coupled to the supporting member.
 12. The system ofclaim 1, wherein the detector comprises a charge-coupled device.
 13. Thesystem of claim 1, wherein the detector comprises an ultravioletdetector.
 14. The system of claim 1, wherein the detector comprises afluorescence detector.
 15. The system of claim 1, wherein the detectorcomprises a semiconductor based photodetector, and wherein the detectoris coupled to the sensor array.
 16. The system of claim 1, wherein theparticle ranges from about 0.05 micron to about 500 microns.
 17. Thesystem of claim 1, wherein a volume of the particle changes whencontacted with the fluid.
 18. The system of claim 1, wherein theparticle comprises a metal oxide particle.
 19. The system of claim 1,wherein the particle comprises a metal quantum particle.
 20. The systemof claim 1, wherein the particle comprises a semiconductor quantumparticle.
 21. The system of claim 1, wherein the particle comprises areceptor molecule coupled to a polymeric resin.
 22. The system of claim1, wherein the particle comprises a receptor molecule coupled to apolymeric resin, and wherein the polymeric resin comprisespolystyrene-polyethylene glycol-divinyl benzene.
 23. The system of claim1, wherein the particle comprises a receptor molecule coupled to apolymeric resin, and wherein the receptor molecule produces the signalin response to the pH of the fluid.
 24. The system of claim 1, whereinthe particle comprises a receptor molecule coupled to a polymeric resin,and wherein the analyte comprises a metal ion, and wherein the receptorproduces the signal in response to the presence of the metal ion. 25.The system of claim 1, wherein the particle comprises a receptormolecule coupled to a polymeric resin, and wherein the analyte comprisesa carbohydrate, and wherein the receptor produces a signal in responseto the presence of a carbohydrate.
 26. The system of claim 1, whereinthe particle comprises a receptor molecule coupled to a polymeric resin,and wherein the particles further comprises a first indicator and asecond indicator, the first and second indicators being coupled to thereceptor, wherein the interaction of the receptor with the analytecauses the first and second indicators to interact such that the signalis produced.
 27. The system of claim 1, wherein the particle comprises areceptor molecule coupled to a polymeric resin, and wherein theparticles further comprises an indicator, wherein the indicator isassociated with the receptor such that in the presence of the analytethe indicator is displaced from the receptor to produce the signal. 28.The system of claim 1, wherein the particle comprises a receptormolecule coupled to a polymeric resin, and wherein the receptorcomprises a polynucleotide.
 29. The system of claim 1, wherein theparticle comprises a receptor molecule coupled to a polymeric resin, andwherein the receptor comprises a peptide.
 30. The system of claim 1,wherein the particle comprises a receptor molecule coupled to apolymeric resin, and wherein the receptor comprises an enzyme.
 31. Thesystem o f claim 1, wherein the particle comprises a receptor moleculecoupled to a polymeric resin, and wherein the receptor comprises asynthetic receptor.
 32. The system of claim 1, wherein the particlecomprises a receptor molecule coupled to a polymeric resin, and whereinthe receptor comprises an unnatural biopolymer.
 33. The system of claim1, wherein the particle comprises a receptor molecule coupled to apolymeric resin, and wherein the receptor comprises an antibody.
 34. Thesystem of claim 1, wherein the particle comprises a receptor moleculecoupled to a polymeric resin, and wherein the receptor comprises anantigen.
 35. The system of claim 1, wherein the analyte comprisesphosphate functional groups, and wherein the particle is configured toproduce the signal in the presence of the phosphate functional groups.36. The system of claim 1, wherein the analyte comprises bacteria, andwherein the particle is configured to produce the signal in the presenceof the bacteria.
 37. The system of claim 1, wherein the system comprisesa plurality of particles positioned within a plurality of cavities, andwherein the plurality of particles produce a detectable pattern in thepresence of the analyte.
 38. The system of claim 1, wherein thesupporting member comprises silicon.
 39. The system of claim 1, whereinthe sensor array further comprises a top cover layer, wherein the topcover layer is coupled to a top surface of the supporting member suchthat the particle is substantially contained within the cavity by thetop cover layer.
 40. The system of claim 1, wherein the sensor arrayfurther comprises a bottom layer coupled to the supporting member, andwherein the bottom layer comprises silicon nitride.
 41. The system ofclaim 1, wherein the particles produce a detectable pattern in thepresence of the analyte.
 42. The system of claim 1, wherein the cavityis configured such that the fluid entering the cavity passes through thesupporting member during use.
 43. The system of claim 1, wherein thelight source comprises a red light emitting diode, a blue light emittingdiode, and a green light emitting diode.
 44. The system of claim 1,wherein the sensor array further comprises a cover layer coupled to thesupporting member and a bottom layer coupled to the supporting member,wherein the cover layer and the bottom layer are removable.
 45. Thesystem of claim 1, wherein the sensor array further comprises a coverlayer coupled to the supporting member and a bottom layer coupled to thesupporting member, wherein the cover layer and the bottom layer areremovable, and wherein the cover layer and the bottom layer includeopenings that are substantially aligned with the cavities during use.46. The system of claim 1, wherein the sensor array further comprises acover layer coupled to the supporting member and a bottom layer coupledto the supporting member, wherein the bottom layer is coupled to abottom surface of the supporting member and wherein the cover layer isremovable, and wherein the cover layer and the bottom layer includeopenings that are substantially aligned with the cavities during use.47. The system of claim 1, wherein the sensor array further comprises acover layer coupled to the supporting member and a bottom layer coupledto the supporting member, wherein an opening is formed in the coverlayer substantially aligned with the cavity, and wherein an opening isformed in the bottom layer substantially aligned with the cavity. 48.The system of claim 1, wherein the cavity is substantially tapered suchthat the width of the cavity narrows in a direction from a top surfaceof the supporting member toward a bottom surface of the supportingmember, and wherein a minimum width of the cavity is substantially lessthan a width of the particle.
 49. The system of claim 1, wherein a widthof a bottom portion of the cavity is substantially less than a width ofa top portion of the cavity, and wherein the width of the bottom portionof the cavity is substantially less than a width of the particle. 50.The system of claim 1, wherein the sensor array further comprises acover layer coupled to the supporting member and a bottom layer coupledto the supporting member, wherein the bottom layer is configured tosupport the particle, and wherein an opening is formed in the coverlayer substantially aligned with the cavity.
 51. The system of claim 1,further comprising a removable cover layer.
 52. The system of claim 1,wherein the supporting member comprises a plastic material.
 53. Thesystem of claim 1, wherein the supporting member comprises a siliconwafer.
 54. The system of claim 1, wherein the supporting membercomprises a dry film photoresist material.
 55. The system of claim 1,wherein the supporting member comprises a plurality of layers of a dryfilm photoresist material.
 56. The system of claim 1, wherein an innersurface of the cavity is coated with a reflective material.
 57. Thesystem of claim 1, further comprising channels in the supporting member,wherein the channels are configured to allow the fluid to flow throughthe channels into and away from the cavity.
 58. The system of claim 1,wherein the sensor array further comprises a pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the cavity, and wherein a channel is formed in the supportingmember, the channel coupling the pump to the cavity such that the fluidflows through the channel to the cavity during use.
 59. The system ofclaim 1, wherein the sensor array further comprises a pump coupled tothe supporting member, wherein the pump is configured to direct thefluid towards the cavity, and wherein a channel is formed in thesupporting member, the channel coupling the pump to the cavity such thatthe fluid flows through the channel to the cavity during use, andwherein the pump comprises a diaphragm pump.
 60. The system of claim 1,wherein the sensor array further comprises a pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the cavity, and wherein a channel is formed in the supportingmember, the channel coupling the pump to the cavity such that the fluidflows through the channel to the cavity during use, and wherein the pumpcomprises a diaphragm pump, and wherein the pump comprises an electrodepump.
 61. The system of claim 1 wherein the sensor array furthercomprises a pump coupled to the supporting member, wherein the pump isconfigured to direct the fluid towards the cavity, and wherein a channelis formed in the supporting member, the channel coupling the pump to thecavity such that the fluid flows through the channel to the cavityduring use, and wherein the pump comprises a diaphragm pump, and whereinthe pump comprises a piezoelectric pump.
 62. The system of claim 1,wherein the sensor array further comprises a pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the cavity, and wherein a channel is formed in the supportingmember, the channel coupling the pump to the cavity such that the fluidflows through the channel to the cavity during use, and wherein the pumpcomprises a diaphragm pump, and wherein the pump comprises a pneumaticactivated pump.
 63. The system of claim 1, wherein the sensor arrayfurther comprises a pump coupled to the supporting member, wherein thepump is configured to direct the fluid towards the cavity, and wherein achannel is formed in the supporting member, the channel coupling thepump to the cavity such that the fluid flows through the channel to thecavity during use, and wherein the pump comprises a diaphragm pump, andwherein the pump comprises a heat activated pump.
 64. The system ofclaim 1, wherein the sensor array further comprises a pump coupled tothe supporting member, wherein the pump is configured to direct thefluid towards the cavity, and wherein a channel is formed in thesupporting member, the channel coupling the pump to the cavity such thatthe fluid flows through the channel to the cavity during use, andwherein the pump comprises a diaphragm pump, and wherein the pumpcomprises a peristaltic pump.
 65. The system of claim 1, wherein thesensor array further comprises a pump coupled to the supporting member,wherein the pump is configured to direct the fluid towards the cavity,and wherein a channel is formed in the supporting member, the channelcoupling the pump to the cavity such that the fluid flows through thechannel to the cavity during use, and wherein the pump comprises adiaphragm pump, and wherein the pump comprises an electroosmosis pump.66. The system of claim 1, wherein the sensor array further comprises apump coupled to the supporting member, wherein the pump is configured todirect the fluid towards the cavity, and wherein a channel is formed inthe supporting member, the channel coupling the pump to the cavity suchthat the fluid flows through the channel to the cavity during use, andwherein the pump comprises a diaphragm pump, and wherein the pumpcomprises an electrohydrodynamic pump.
 67. The system of claim 1,wherein the sensor array further comprises a pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the cavity, and wherein a channel is formed in the supportingmember, the channel coupling the pump to the cavity such that the fluidflows through the channel to the cavity during use, and wherein the pumpcomprises a diaphragm pump, and wherein the pump comprises anelectroosmosis pump and an electrohydrodynamic pump.
 68. The system ofclaim 1, wherein the particle comprises a receptor molecule coupled to apolymeric resin, and wherein the particle further comprises a firstindicator and a second indicator, the first and second indicators beingcoupled to the receptor, wherein the interaction of the receptor withthe analyte causes the first and second indicators to interact such thatthe signal is produced.
 69. The system of claim 1, wherein the particlecomprises a receptor molecule coupled to a polymeric resin, and whereinthe particle further comprises an indicator, wherein the indicator isassociated with the receptor such that in the presence of the analytethe indicator is displaced from the receptor to produce the signal. 70.The system of claim 1, wherein a portion of the supporting member issubstantially transparent to a portion of light produced by the lightsource.
 71. The system of claim 1, wherein the particle is coupled tothe supporting member with via an adhesive material.
 72. The system ofclaim 1, wherein the particle are coupled to the supporting member via agel material.
 73. The system of claim 1, wherein the particle issuspended in a gel material, the gel material covering a portion of thesupporting member, and wherein a portion of the particle extends fromthe upper surface of the gel.
 74. The system of claim 1, wherein thesensor array further comprises a cover coupled to the supporting member,positioned above the particle, wherein a force exerted by the cover onthe particle inhibits the displacement of the particle from thesupporting member.
 75. The system of claim 1, wherein the supportingmember comprises glass.
 76. The system of claim 1, wherein thesupporting member is composed of a material substantially transparent toultraviolet light.
 77. The system of claim 1, further comprising aconduit coupled to the sensor array, wherein the conduit is configuredto conduct the fluid sample to and away from the sensor array; and avacuum chamber coupled to the conduit, wherein the vacuum chambercomprises a breakable barrier positioned between the chamber and theconduit, and wherein the chamber is configured to pull the fluid throughthe conduit when the breakable barrier is punctured.
 78. The system ofclaim 1, further comprising a conduit coupled to the sensor array,wherein the conduit is configured to conduct the fluid sample to andaway from the sensor array; and a vacuum chamber coupled to the conduit,wherein the vacuum chamber comprises a breakable barrier positionedbetween the chamber and the conduit, and wherein the chamber isconfigured to pull the fluid through the conduit when the breakablebarrier is punctured, and further comprising a filter coupled to theconduit and the sensor array, wherein the fluid passes through thefilter before reaching the sensor array.
 79. The system of claim 1,further comprising a conduit coupled to the sensor array, wherein theconduit is configured to conduct the fluid sample to and away from thesensor array; and a vacuum chamber coupled to the conduit, wherein thevacuum chamber comprises a breakable barrier positioned between thechamber and the conduit, and wherein the chamber is configured to pullthe fluid through the conduit when the breakable barrier is punctured,and further comprising a filter coupled to the conduit and the sensorarray, wherein the fluid passes through the filter before reaching thesensor array, and wherein the fluid is a blood sample, and wherein thefilter comprises a membrane for the removal of particulates.
 80. Thesystem of claim 1, further comprising a conduit coupled to the sensorarray, wherein the conduit is configured to conduct the fluid sample toand away from the sensor array; and a vacuum chamber coupled to theconduit, wherein the vacuum chamber comprises a breakable barrierpositioned between the chamber and the conduit, and wherein the chamberis configured to pull the fluid through the conduit when the breakablebarrier is punctured, and further comprising a filter coupled to theconduit and the sensor array, wherein the fluid passes through thefilter before reaching the sensor array, and wherein the fluid is ablood sample, and wherein the filter comprises a membrane for removal ofwhite and red blood cells from the blood.
 81. The system of claim 1,wherein the particle comprises a biopolymer coupled to a polymericresin, and wherein the biopolymer undergoes a chemical reaction in thepresence of the analyte to produce a signal.
 82. The system of claim 1,wherein the particle comprises a biopolymer coupled to a polymericresin, and wherein the biopolymer undergoes a chemical reaction in thepresence of the analyte to produce a signal, and wherein the chemicalreaction comprises cleavage of the biopolymer by the analyte.
 83. Thesystem of claim 1, wherein the particle comprises a biopolymer coupledto a polymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe biopolymer comprises a peptide, and wherein the analyte comprises aprotease, and wherein the chemical reaction comprises cleavage of thepeptide by the protease.
 84. The system of claim 1, wherein the particlecomprises a biopolymer coupled to a polymeric resin, and wherein thebiopolymer undergoes a chemical reaction in the presence of the analyteto produce a signal, and wherein the biopolymer comprises apolynucleotide, and wherein the analyte comprises a nuclease, andwherein the chemical reaction comprises cleavage of the polynucleotideby the nuclease.
 85. The system of claim 1, wherein the particlecomprises a biopolymer coupled to a polymeric resin, and wherein thebiopolymer undergoes a chemical reaction in the presence of the analyteto produce a signal, and wherein the biopolymer comprises anoligosaccharide, and wherein the analyte comprises an oligosaccharidecleaving agent, and wherein the chemical reaction comprises cleavage ofthe oligosaccharide by the oligosaccharide cleaving agent.
 86. Thesystem of claim 1, wherein the particle comprises a biopolymer coupledto a polymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe particle further comprises a first indicator and a second indicator,the first an d second indicators being coup led to the biopolymer, andwherein the chemical reaction of the biopolymer in the presence of theanalyte causes a distance between the first and second indicators tobecome altered such that the signal is produced.
 87. The system of claim1, wherein the particle comprises a biopolymer coupled to a polymericresin, and wherein the biopolymer undergoes a chemical reaction in thepresence of the analyte to produce a signal, and wherein the particlefurther comprises a first indicator and a second indicator, the firstand second indicators being coupled to the biopolymer, and wherein thechemical reaction of the biopolymer in the presence of the analytecauses a distance between the first and second indicators to becomealtered such that the signal is produced, and wherein the firstindicator is a fluorescent dye and wherein the second indicator is afluorescent quencher, and wherein the first indicator and the secondindicator are within the Foster energy transfer radius, and wherein thechemical reaction of the biopolymer in the presence of the analytecauses the first and second indicators to move outside the Foster energytransfer radius.
 88. The system of claim 1, wherein the particlecomprises a biopolymer coupled to a polymeric resin, and wherein thebiopolymer undergoes a chemical reaction in the presence of the analyteto produce a signal, and wherein the particle further comprises a firstindicator and a second indicator, the first and second indicators beingcoupled to the biopolymer, and wherein the chemical reaction of thebiopolymer in the presence of the analyte causes a distance between thefirst and second indicators to become altered such that the signal isproduced, wherein the first indicator is a fluorescent dye and whereinthe second indicator is a different fluorescent dye, and wherein thefirst indicator and the second indicator produce a fluorescenceresonance energy transfer signal, and wherein the chemical reaction ofthe biopolymer in the presence of the analyte causes the positions ofthe first and second indicators to change such that the fluorescenceresonance energy transfer signal is altered.
 89. The system of claim 1,wherein the particle comprises a biopolymer coupled to a polymericresin, and wherein the biopolymer undergoes a chemical reaction in thepresence of the analyte to produce a signal, and further comprising anindicator coupled to the biopolymer, and wherein the chemical reactionof the biopolymer in the presence of the analyte causes the biopolymerto be cleaved such that a portion of the biopolymer coupled to theindicator is cleaved from a portion of the biopolymer coupled to thepolymeric resin.
 90. The system of claim 1 wherein the particlecomprises a biopolymer coupled to a polymeric resin, and wherein thebiopolymer undergoes a chemical reaction in the presence of the analyteto produce a signal, and wherein the particle further comprises anindicator coupled to the particle, and wherein the chemical reactioncauses a change to a biopolymer such that the interaction of theindicator with the biopolymer is altered to produce the signal.
 91. Thesystem of claim 1, wherein the particle comprises a biopolymer coupledto a polymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe particle further comprises an indicator coupled to the particle, andwherein the chemical reaction causes a change to the biopolymer and theindicator to produce the signal.
 92. The system of claim 1, wherein theparticle comprises a receptor coupled to a polymeric resin, and a probemolecule coupled to the polymeric resin, and wherein the probe moleculeis configured to produce a signal when the receptor interacts with theanalyte during use.
 93. The system of claim 1, wherein the particlecomprises a receptor coupled to a polymeric resin, and a probe moleculecoupled to the polymeric resin, and wherein the probe molecule isconfigured to produce a signal when the receptor interacts with theanalyte during use, and wherein the particles further comprises anadditional probe molecule coupled to the polymeric resin, wherein theinteraction of the receptor with the analyte causes the probe moleculesto interact such that the signal is produced.
 94. A system for detectingan analyte in a fluid comprising: a light source; a sensor array, thesensor array comprising: a supporting member; wherein a first cavity anda second cavity are formed within the supporting member; a firstparticle positioned within the first cavity; a second particlepositioned within the second cavity, wherein the second particlecomprises a reagent, wherein a portion of the reagent is removable fromthe second particle when contacted with a decoupling solution, andwherein the reagent is configured to modify the first particle, when thereagent is contacted with the first particle, such that the firstparticle will produce a signal when the first particle interacts withthe analyte during use; a first pump coupled to the supporting member,wherein the pump is configured to direct the fluid towards the firstcavity; a second pump coupled to the supporting member, wherein thesecond pump is configured to direct the decoupling solution towards thesecond cavity; wherein a first channel is formed in the supportingmember, the first channel coupling the first pump to the first cavitysuch that the fluid flows through the first channel to the first cavityduring use, and wherein a second channel is formed in the supportingmember, the second channel coupling the second cavity to the firstcavity such that the decoupling solution flows from the second cavitythrough the second channel to the first cavity during use; and adetector, the detector being configured to detect the signal produced bythe interaction of the analyte with the particle during use; wherein thelight source and detector are positioned such that light passes from thelight source, to the particle, and onto the detector during use.
 95. Thesystem of claim 94, wherein the sensor array further comprises aplurality of additional particles positioned within a plurality ofadditional cavities, and wherein the system is configured tosubstantially simultaneously detect a plurality of analytes in thefluid, and wherein the second cavity is coupled to the additionalcavities such that the reagent may be transferred from the secondparticle to the additional cavities during use.
 96. The system of claim94, wherein the first particle comprises an indicator molecule coupledto a first polymeric resin, and the second particle comprises a receptormolecule coupled to a second polymeric resin.
 97. The system of claim94, wherein the first particle comprises a first polymeric resinconfigured to bind to the receptor molecule, and wherein the secondparticle comprises the receptor molecule coupled to a second polymericresin.
 98. The system of claim 94, wherein the sensor array furthercomprises a reservoir coupled to the second pump, the reservoirconfigured to hold the decoupling solution.
 99. A system for detectingan analyte in a fluid comprising: a light source; a sensor array, thesensor array comprising at least one particle coupled to the sensorarray, wherein the particle is configured to produce a signal when theparticle interacts with the analyte; and a detector configured to detectthe signal produced by the interaction of the analyte with the particle;wherein the light source and detector are positioned such that lightpasses from the light source, to the particle, and onto the detectorduring use.
 100. A sensor array for detecting an analyte in a fluidcomprising: a supporting member; wherein at least one cavity is formedwithin the supporting member; a particle positioned within the cavity,wherein the particle is configured to produce a signal when the particleinteracts with the analyte.
 101. The sensor array of claim 100, furthercomprising a plurality of particles positioned within the cavity. 102.The sensor array of claim 100, wherein the particle comprises a receptormolecule coupled to a polymeric resin.
 103. The sensor array of claim100, wherein the particle has a size ranging from about 0.05 micron toabout 500 microns in diameter.
 104. The sensor array of claim 100,wherein the particle has a size ranging from about 0.05 micron to about500 microns in diameter, and wherein the cavity is configured tosubstantially contain the particle.
 105. The sensor array of claim 100,wherein the supporting member comprises a plastic material.
 106. Thesensor array of claim 100, wherein the supporting member comprises asilicon wafer.
 107. The sensor array of claim 100, wherein the cavityextends through the supporting member.
 108. The sensor array of claim100, wherein the supporting member comprises a silicon wafer, andwherein the cavity is substantially pyramidal in shape and wherein thesidewalls of the cavity are substantially tapered at an angle of betweenabout 50 to about 60 degrees.
 109. The sensor array of claim 100,wherein the supporting member comprises a silicon wafer, and furthercomprising a substantially transparent layer positioned on a bottomsurface of the silicon wafer.
 110. The sensor array of claim 100,wherein the supporting member comprises a silicon wafer, and furthercomprising a substantially transparent layer positioned on a bottomsurface of the silicon wafer, wherein the substantially transparentlayer comprises silicon dioxide, silicon nitride, or siliconoxide/silicon nitride multilayer stacks.
 111. The sensor array of claim100, wherein the supporting member comprises a silicon wafer, andfurther comprising a substantially transparent layer positioned on abottom surface of the silicon wafer, wherein the substantiallytransparent layer comprises silicon nitride.
 112. The sensor array ofclaim 100, wherein the supporting member comprises a silicon wafer, andwherein the silicon wafer has an area of about 1 cm² to about 100 cm².113. The sensor array of claim 100, further comprising a plurality ofcavities formed in the silicon wafer, and wherein from about 10 to about10⁶ cavities are formed in the silicon wafer.
 114. The sensor array ofclaim 100, further comprising channels in the supporting member, whereinthe channels are configured to allow the fluid to flow through thechannels into and away from the cavity.
 115. The sensor array of claim100, further comprising an inner surface coating, wherein the innersurface coating is configured to inhibit dislodgment of the particle.116. The sensor array of claim 100, further comprising a detectorcoupled to the bottom surface of the supporting member, wherein thedetector is positioned below the cavity.
 117. The sensor array of claim100, further comprising a detector coupled to the bottom surface of thesupporting member, wherein the detector is positioned below the cavity,and wherein the detector is a semiconductor based photodetector. 118.The sensor array of claim 100, further comprising a detector coupled tothe bottom surface of the supporting member, wherein the detector ispositioned below the cavity, and wherein the detector is a Fabry-Perottype detector.
 119. The sensor array of claim 100, further comprising adetector coupled to the bottom surface of the supporting member, whereinthe detector is positioned below the cavity, and further comprising anoptical fiber coupled to the detector, wherein the optical fiber isconfigured to transmit optical data to a microprocessor.
 120. The sensorarray of claim 100, further comprising an optical filters coupled to abottom surface of the sensor array.
 121. The sensor array of claim 100,further comprising a barrier layer positioned over the cavity, thebarrier layer being configured to inhibit dislodgment of the particleduring use.
 122. The sensor array of claim 100, further comprising abarrier layer positioned over the cavity, the barrier layer beingconfigured to inhibit dislodgment of the particle during use, andwherein the barrier layer comprises a substantially transparent coverplate positioned over the cavity, and wherein the cover plate ispositioned a fixed distance over the cavity such that the fluid canenter the cavity.
 123. The sensor array of claim 100, further comprisinga barrier layer positioned over the cavity, the barrier layer beingconfigured to inhibit dislodgment of the particle during use, andwherein the barrier layer comprises a substantially transparent coverplate positioned over the cavity, and wherein the cover plate ispositioned a fixed distance over the cavity such that the fluid canenter the cavity, and wherein the barrier layer comprises plastic,glass, quartz, silicon oxide, or silicon nitride.
 124. The sensor arrayof claim 100, further comprising a plurality of particles positionedwithin a plurality of cavities formed in the supporting member.
 125. Thesensor array of claim 100, wherein the system comprises a plurality ofparticles positioned within a plurality of cavities, and wherein theplurality of particles produce a detectable pattern in the presence ofthe analyte.
 126. The sensor array of claim 100, further comprisingchannels in the supporting member, wherein the channels are configuredto allow the fluid to flow through the channels into and away from thecavities, and wherein the barrier layer comprises a cover platepositioned upon an upper surface of the supporting member, and whereinthe cover plate inhibits passage of the fluid into the cavities suchthat the fluid enters the cavities via the channels.
 127. The sensorarray of claim 100, further comprising a cover layer coupled to thesupporting member and a bottom layer coupled to the supporting member,wherein the bottom layer is coupled to a bottom surface of thesupporting member and wherein the cover layer is removable, and whereinthe cover layer and the bottom layer include openings that aresubstantially aligned with the cavities during use.
 128. The sensorarray of claim 100, further comprising a cover layer coupled to thesupporting member and a bottom layer coupled to the supporting member,wherein an opening is formed in the cover layer substantially alignedwith the cavity, and wherein an opening is formed in the bottom layersubstantially aligned with the cavity.
 129. The sensor array of claim100, wherein the cavity is substantially tapered such that the width ofthe cavity narrows in a direction from a top surface of the supportingmember toward a bottom surface of the supporting member, and wherein aminimum width of the cavity is substantially less than a width of theparticle.
 130. The sensor array of claim 100, wherein a width of abottom portion of the cavity is substantially less than a width of a topportion of the cavity, and wherein the width of the bottom portion ofthe cavity is substantially less than a width of the particle.
 131. Thesensor array of claim 100, further comprising a cover layer coupled tothe supporting member and a bottom layer coupled to the supportingmember, wherein the bottom layer is configured to support the particle,and wherein an opening is formed in the cover layer substantiallyaligned with the cavity.
 132. The sensor array of claim 100, furthercomprising a removable cover layer coupled to the supporting member.133. The sensor array of claim 100, wherein the supporting membercomprises a dry film photoresist material.
 134. The sensor array ofclaim 100, wherein the supporting member comprises a plurality of layersof a dry film photoresist material.
 135. The sensor array of claim 100,wherein an inner surface of the cavity is coated with a reflectivematerial.
 136. The sensor array of claim 100, further comprisingchannels in the supporting member, wherein the channels are configuredto allow the fluid to flow through the channels into and away from thecavity.
 137. The sensor array of claim 100, further comprising a pumpcoupled to the supporting member, wherein the pump is configured todirect the fluid towards the cavity; and a channel formed in thesupporting member, the channel coupling the pump to the cavity such thatthe fluid flows through the channel to the cavity during use.
 138. Thesensor array of claim 100, further comprising a pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the cavity; and a channel formed in the supporting member, thechannel coupling the pump to the cavity such that the fluid flowsthrough the channel to the cavity during use, and wherein the pumpcomprises a diaphragm pump.
 139. The sensor array of claim 100, furthercomprising a pump coupled to the supporting member, wherein the pump isconfigured to direct the fluid towards the cavity; and a channel formedin the supporting member, the channel coupling the pump to the cavitysuch that the fluid flows through the channel to the cavity during use,and wherein the pump comprises an electrode pump.
 140. The sensor arrayof claim 100, further comprising a pump coupled to the supportingmember, wherein the pump is configured to direct the fluid towards thecavity; and a channel formed in the supporting member, the channelcoupling the pump to the cavity such that the fluid flows through thechannel to the cavity during use, and wherein the pump comprises apiezoelectric pump.
 141. The sensor array of claim 100, furthercomprising a pump coupled to the supporting member, wherein the pump isconfigured to direct the fluid towards the cavity; and a channel formedin the supporting member, the channel coupling the pump to the cavitysuch that the fluid flows through the channel to the cavity during use,and wherein the pump comprises a pneumatic activated pump.
 142. Thesensor array of claim 100, further comprising a pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the cavity; and a channel formed in the supporting member, thechannel coupling the pump to the cavity such that the fluid flowsthrough the channel to the cavity during use, and wherein the pumpcomprises a heat activated pump.
 143. The sensor array of claim 100,further comprising a pump coupled to the supporting member, wherein thepump is configured to direct the fluid towards the cavity; and a channelformed in the supporting member, the channel coupling the pump to thecavity such that the fluid flows through the channel to the cavityduring use, and wherein the pump comprises a peristaltic pump.
 144. Thesensor array of claim 100, further comprising a pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the cavity; and a channel formed in the supporting member, thechannel coupling the pump to the cavity such that the fluid flowsthrough the channel to the cavity during use, and wherein the pumpcomprises an electroosmosis pump.
 145. The sensor array of claim 100,further comprising a pump coupled to the supporting member, wherein thepump is configured to direct the fluid towards the cavity; and a channelformed in the supporting member, the channel coupling the pump to thecavity such that the fluid flows through the channel to the cavityduring use, and wherein the pump comprises an electrohydrodynamic pump.146. The sensor array of claim 100, further comprising a pump coupled tothe supporting member, wherein the pump is configured to direct thefluid towards the cavity; and a channel formed in the supporting member,the channel coupling the pump to the cavity such that the fluid flowsthrough the channel to the cavity during use, and wherein the pumpcomprises an electroosmosis pump and an electrohydrodynamic pump. 147.The sensor array of claim 100, wherein a width of a bottom portion ofthe cavity is substantially less than a width of a top portion of thecavity, and wherein the width of the bottom portion of the cavity issubstantially less than a width of the particle.
 148. A sensor array fordetecting an analyte in a fluid comprising: a supporting member; whereina first cavity and a second cavity are formed within the supportingmember; a first particle positioned within the first cavity; a secondparticle positioned within the second cavity, wherein the secondparticle comprises a reagent, wherein a portion of the reagent isremovable from the second particle when contacted with a decouplingsolution, and wherein the reagent is configured to modify the firstparticle, when the reagent is contacted with the first particle, suchthat the first particle will produce a signal when the first particleinteracts with the analyte during use; a first pump coupled to thesupporting member, wherein the pump is configured to direct the fluidtowards the first cavity; a second pump coupled to the supportingmember, wherein the second pump is configured to direct the decouplingsolution towards the second cavity; wherein a first channel is formed inthe supporting member, the first channel coupling the first pump to thefirst cavity such that the fluid flows through the first channel to thefirst cavity during use, and wherein a second channel is formed in thesupporting member, the second channel coupling the second cavity to thefirst cavity such that the decoupling solution flows from the secondcavity through the second channel to the first cavity during use. 149.The sensor array of claim 148, wherein the first particle comprises areceptor molecule coupled to a first polymeric resin, and wherein thesecond particle comprises an indicator molecule coupled to a secondpolymeric resin.
 150. The sensor array of claim 148, wherein the firstparticle comprises an indicator molecule coupled to a first polymericresin, and the second particle comprises a receptor molecule coupled toa second polymeric resin.
 151. The sensor array of claim 148, whereinthe first particle comprises a first polymeric resin configured to bindto the receptor molecule, and wherein the second particle comprises thereceptor molecule coupled to a second polymeric resin.
 152. The sensorarray of claim 148, further comprising a reservoir coupled to the secondpump, the reservoir configured to hold the decoupling solution.
 153. Asensor array for detecting an analyte in a fluid comprising: at leastone particle coupled to a supporting member, wherein the particle isconfigured to produce a signal when the particle interacts with theanalyte.
 154. The sensor array of claim 153, wherein the particle iscoupled to the supporting member with via an adhesive material.
 155. Thesensor array of claim 153, wherein the particle are coupled to thesupporting member via a gel material.
 156. The sensor array of claim153, wherein the particle is suspended in a gel material, the gelmaterial covering a portion of the supporting member, and wherein aportion of the particle extends from the upper surface of the gel. 157.The sensor array of claim 153, further comprising a cover positionedabove the particle.
 158. The sensor array of claim 153, furthercomprising a cover coupled to the supporting member, positioned abovethe particle, wherein a force exerted by the cover on the particleinhibits the displacement of the particle from the supporting member.159. The sensor array of claim 153, wherein the particle comprises areceptor molecule coupled to a polymeric resin.
 160. The sensor array ofclaim 153, wherein the supporting member comprises glass.
 161. A methodfor forming a sensor array configured to detect an analyte in a fluid,comprising: forming a cavity in a supporting member, wherein thesupporting member comprises a silicon wafer; placing a particle in thecavity, wherein the particle is configured to produce a signal when theparticle interacts with the analyte; and forming a cover upon a portionof the supporting member, wherein the cover is configured to inhibitdislodgment of the particle from the cavity.
 162. The method of claim161, wherein forming the cavity comprises anisotropically etching thesilicon wafer.
 163. The method of claim 161, wherein forming the cavitycomprises anisotropically etching the silicon wafer with a wet hydroxideetch.
 164. The method of claim 161, wherein forming the cavity comprisesanisotropically etching the silicon wafer such that sidewalls of thecavity are tapered at an angle from about 50 degrees to about 60degrees.
 165. The method of claim 161, wherein the silicon wafer has anarea of about 1 cm² to about 100 cm².
 166. The method of claim 161,further comprising forming a substantially transparent layer upon abottom surface of the silicon wafer below the cavity.
 167. The method ofclaim 161, further comprising forming a substantially transparent layerupon a bottom surface of the silicon wafer, wherein the cavity extendsthrough the silicon wafer and wherein the substantially transparentlayer is positioned to support the particle.
 168. The method of claim161, wherein the substantially transparent layer comprises siliconnitride.
 169. The method of claim 161, wherein the cover comprisesplastic, glass, quartz, silicon nitride, or silicon oxide.
 170. Themethod of claim 161, wherein forming the cover comprises coupling thecover to the silicon wafer at a distance above the silicon wafersubstantially less than a width of the particle.
 171. The method ofclaim 161, further comprising etching channels in the silicon waferprior to forming the cover on the silicon wafer, wherein forming thecover comprises placing the cover against the upper surface of thesilicon wafer, and wherein the channels are configured to allow thefluid to pass through the silicon wafer to and from the cavities. 172.The method of claim 161, further comprising coating an inner surface ofthe cavity with a material to increase adhesion of the particle to theinner surface of the cavity.
 173. The method of claim 161, furthercomprising coating an inner surface of the cavity with a material toincrease reflectivity of the inner surface of the cavity.
 174. Themethod of claim 161, further comprising forming an optical detector upona bottom surface of the supporting member below the cavity.
 175. Themethod of claim 161, further comprising forming a sensing cavity upon abottom surface of the supporting member below the cavity.
 176. Themethod of claim 161, further comprising forming a sensing cavity upon abottom surface of the supporting member below the cavity, and whereinforming the sensing cavity comprises: forming a barrier layer upon abottom surface of the silicon wafer; forming a bottom diaphragm layerupon the barrier layer; forming etch windows extending through thebottom diaphragm layer; forming a sacrificial spacer layer upon thebottom diaphragm layer; removing a portion of the spacer layer; forminga top diaphragm layer; and removing a remaining portion of the spacerlayer.
 177. The method of claim 161, further comprising forming anoptical filter upon the bottom surface of the supporting member. 178.The method of claim 161, further comprising forming a plurality ofcavities in the silicon wafer.
 179. The method of claim 161, whereinfrom about 10 to about 10⁶ cavities are formed in the silicon wafer.180. The method of claim 161, wherein the formed cavity is configured toallow the fluid to pass through the supporting member.
 181. The methodof claim 161, further comprising forming a substantially transparentlayer upon a bottom surface of the supporting member below the cavity,wherein the bottom layer is configured to inhibit the displacement ofthe particle from the cavity while allowing the fluid to pass throughthe supporting member.
 182. The system of claim 161, wherein a width ofa bottom portion of the cavity is substantially less than a width of atop portion of the cavity, and wherein the width of the bottom portionof the cavity is substantially less than a width of the particle. 183.The method of claim 161, further comprising forming channels in thesupporting member wherein the channels are configured to allow the fluidto pass through the supporting member to and from the cavity.
 184. Themethod of claim 161, further comprising forming a pump on the supportingmember, the pump being configured to pump the fluid to the cavity. 185.The method of claim 161, further comprising forming a cover, whereinforming the cover comprises: forming a removable layer upon the uppersurface of the supporting member; forming a cover upon the removablelayer; forming support structures upon the supporting member, thesupport structures covering a portion of the cover; and dissolving theremovable layer.
 186. The method of claim 161, wherein forming the coverfurther comprises forming openings in the cover, wherein the openingsare substantially aligned with the cavity.
 187. The method of claim 161,wherein the particles are placed in the cavities using amicromanipulator.
 188. The method of claim 161, further comprisingforming additional cavities within the supporting member, and furthercomprising placing additional particles in the additional cavities,wherein placing the additional particles in the additional cavitiescomprises: placing a first masking layer on the supporting member,wherein the first masking layer covers a first portion of the additionalcavities such that passage of a particle into the first portion of theadditional cavities is inhibited, and wherein the first masking layer asecond portion of the cavities substantially unmasked,; placing theadditional particles on the supporting member; moving the additionalparticles across the supporting member such that the particles fall intothe second portion of the cavities; removing the first masking layer;placing a second masking layer upon the supporting member, wherein thesecond masking layer covers the second portion of the cavities and aportion of the first portion of the cavities while leaving a thirdportion of the cavities unmasked; placing additional particles on thesupporting member; and moving the additional particles across thesupporting member such that the particle fall into the third portion ofthe cavities.
 189. The method of claim 161, wherein forming the covercomprises coupling the cover to the supporting member at a distanceabove the supporting member substantially less than a width of theparticle.
 190. The method of claim 161, wherein the supporting membercomprises a dry film photoresist material.
 191. The method of claim 161,wherein the supporting member comprises a plurality of layers of a dryfilm photoresist material.
 192. The method of claim 161, wherein formingthe cavity comprises: etching a first opening through a first dry filmphotoresist layer, the first opening having a width substantially lessthan a width of the particle; placing a second dry film photoresistlayer upon the first dry film photoresist layer; etching a secondopening through the second dry film photoresist layer, the secondopening being substantially aligned with the first opening, wherein awidth of the second opening is substantially greater than the width ofthe first opening; wherein the second dry film photoresist layercomprises a thickness substantially greater than a width of theparticle; and further comprising forming a reflective layer upon theinner surface of the cavity.
 193. The method of claim 161, wherein thesupporting material comprises a plastic material.
 194. The method ofclaim 161, wherein the supporting material comprises a plastic material,and wherein the cavity is formed by drilling the supporting material.195. The method of claim 161, wherein the supporting material comprisesa plastic material, and wherein the cavity is formed by transfer moldingthe supporting member.
 196. The method of claim 161, wherein thesupporting material comprises a plastic material, and wherein the cavityis formed by a punching device.
 197. A method of sensing an analyte in afluid comprising: passing a fluid over a sensor array, the sensor arraycomprising at least one particle positioned within a cavity of asupporting member; monitoring a spectroscopic change of the particle asthe fluid is passed over the sensor array, wherein the spectroscopicchange is caused by the interaction of the analyte with the particle.198. The method of claim 197, wherein the spectroscopic change comprisesa change in absorbance of the particle.
 199. The method of claim 197,wherein the spectroscopic change comprises a change in fluorescence ofthe particle.
 200. The method of claim 197, wherein the spectroscopicchange comprises a change in phosphorescence of the particle.
 201. Themethod of claim 197, wherein the analyte is a proton atom, and whereinthe spectroscopic change is produced when the pH of the fluid is varied,and wherein monitoring the spectroscopic change of the particle allowsthe pH of the fluid to be determined.
 202. The method of claim 197,wherein the analyte is a metal cation, and wherein the spectroscopicchange is produced in response to the presence of the metal cation inthe fluid.
 203. The method of claim 197, wherein the analyte is ananion, and wherein the spectroscopic change is produced in response tothe presence of the anion in the fluid.
 204. The method of claim 197,wherein the analyte is a DNA molecule, and wherein the spectroscopicchange is produced in response to the presence of the DNA molecule inthe fluid.
 205. The method of claim 197, wherein the analyte is aprotein, and wherein the spectroscopic change is produced in response tothe presence of the protein in the fluid.
 206. The method of claim 197,wherein the analyte is a metabolite, and wherein the spectroscopicchange is produced in response to the presence of the metabolite in thefluid.
 207. The method of claim 197, wherein the analyte is a sugar, andwherein the spectroscopic change is produced in response to the presenceof the sugar in the fluid.
 208. The method of claim 197, wherein theanalyte is a bacteria, and wherein the spectroscopic change is producedin response to the presence of the bacteria in the fluid.
 209. Themethod of claim 197, wherein the particle comprises a receptor coupledto a polymeric resin, and further comprising exposing the particle to anindicator prior to passing the fluid over the sensor array.
 210. Themethod of claim 197, wherein the particle comprises a receptor coupledto a polymeric resin, and further comprising exposing the particle to anindicator prior to passing the fluid over the sensor array, and whereina binding strength of the indicator to the receptor is less than abinding strength of the analyte to the receptor.
 211. The method ofclaim 197, wherein the particle comprises a receptor coupled to apolymeric resin, and further comprising exposing the particle to anindicator prior to passing the fluid over the sensor array, and whereinthe indicator is a fluorescent indicator.
 212. The method of claim 197,further comprising treating the fluid with an indicator prior to passingthe fluid over the sensor array, wherein the indicator is configured tocouple with the analyte.
 213. The method of claim 197, wherein theanalyte is bacteria and further comprising breaking down the bacteriaprior to passing the fluid over the sensor array.
 214. The method ofclaim 197, wherein monitoring the spectroscopic change is performed witha CCD device.
 215. The method of claim 197, further comprising measuringthe intensity of the spectroscopic change, and further comprisingcalculating the concentration of the analyte based on the intensity ofthe spectroscopic change.
 216. The method of claim 197, wherein thesupporting member comprises silicon.
 217. The method of claim 197,wherein the spectroscopic change comprises a change in reflectance ofthe particle.
 218. The method of claim 197, wherein the cavity isconfigured such that the fluid entering the cavity passes through thesupporting member.
 219. The method of claim 197, wherein monitoring thespectroscopic change comprises: directing a red light source at theparticle; detecting the absorbance of red light by the particle;directing a green light source at the particle; detecting the absorbanceof green light by the particle; directing a blue light source at theparticle; and detecting the absorbance of blue light by the particle.220. The method of claim 197, wherein the sensor array further comprisesa vacuum chamber coupled to a conduit and the sensor array, and whereinthe chamber is configured to provide a pulling force on the fluid in thesensor array.
 221. The method of claim 197, wherein the fluid is blood.222. The method of claim 197, further comprising passing the fluidthrough a filter prior to passing the fluid over the sensor array. 223.The method of claim 197, further comprising passing the fluid through areagent reservoir prior to passing the fluid over the sensor array. 224.The method of claim 197, wherein the particles are initially stored in abuffer, and further comprising removing the buffer prior to passing thefluid over the sensor array.
 225. The method of claim 197, wherein theparticle comprises a polymeric resin, a biopolymer coupled to thepolymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal.
 226. Themethod of claim 197, wherein the particle comprises a polymeric resin, abiopolymer coupled to the polymeric resin, and wherein the biopolymerundergoes a chemical reaction in the presence of the analyte to producea signal, and wherein the signal comprises an absorbance of theparticle.
 227. The method of claim 197, wherein the particle comprises apolymeric resin, a biopolymer coupled to the polymeric resin, andwherein the biopolymer undergoes a chemical reaction in the presence ofthe analyte to produce a signal, and wherein the signal comprises afluorescence of the particle.
 228. The method of claim 197, wherein theparticle comprises a polymeric resin, a biopolymer coupled to thepolymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe signal comprises a phosphorescence of the particle.
 229. The methodof claim 197, wherein the particle comprises a polymeric resin, abiopolymer coupled to the polymeric resin, and wherein the biopolymerundergoes a chemical reaction in the presence of the analyte to producea signal, and wherein the chemical reaction comprises cleavage of thebiopolymer induced by the analyte.
 230. The method of claim 197, whereinthe particle comprises a polymeric resin, a biopolymer coupled to thepolymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe biopolymer comprises a peptide, and wherein the analyte comprises aprotease, and wherein the chemical reaction comprises cleavage of thepeptide b y the protease.
 231. The method of claim 197, wherein theparticle comprises a polymeric resin, a biopolymer coupled to thepolymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe biopolymer comprises a polynucleotide, and wherein the analytecomprises a nuclease, and wherein the chemical reaction comprisescleavage of the polynucleotide by the nuclease.
 232. The method of claim197, wherein the particle comprises a polymeric resin, a biopolymercoupled to the polymeric resin, and wherein the biopolymer undergoes achemical reaction in the presence of the analyte to produce a signal,and wherein the biopolymer comprises an oligosaccharide, and wherein theanalyte comprises an oligosaccharide cleaving agent, and wherein thechemical reaction comprises cleavage of the oligosaccharide by theoligosaccharide cleaving agent.
 233. The method of claim 197, whereinthe particle comprises a polymeric resin, a biopolymer coupled to thepolymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe particle further comprises a first indicator and a second indicator,the first and second indicators being coupled to the biopolymer, andwherein the chemical reaction of the biopolymer in the presence of theanalyte causes a distance between the first and second indicators tobecome altered such that the alteration of the signal is produced. 234.The method of claim 197, wherein the particle comprises a polymericresin, a biopolymer coupled to the polymeric resin, and wherein thebiopolymer undergoes a chemical reaction in the presence of the analyteto produce a signal, and wherein the particle further comprises a firstindicator and a second indicator, the first and second indicators beingcoupled to the biopolymer, and wherein the chemical reaction of thebiopolymer in the presence of the analyte causes a distance between thefirst and second indicators to become altered such that the alterationof the signal is produced, and wherein the first indicator is afluorescent dye and wherein the second indicator is a fluorescentquencher, and wherein the first indicator and the second indicator arewithin the Foster energy transfer radius, and wherein the chemicalreaction of the biopolymer in the presence of the analyte causes thefirst and second indicators to move outside the Foster energy transferradius such that the alteration of the signal is produced.
 235. Themethod of claim 197, wherein the particle comprises a polymeric resin, abiopolymer coupled to the polymeric resin, and wherein the biopolymerundergoes a chemical reaction in the presence of the analyte to producea signal, and wherein the particle further comprises a first indicatorand a second indicator, the first and second indicators being coupled tothe biopolymer, and wherein the chemical reaction of the biopolymer inthe presence of the analyte causes a distance between the first andsecond indicators to become altered such that the alteration of thesignal is produced, and wherein the first indicator is a fluorescent dyeand wherein the second indicator is a different fluorescent dye, andwherein the first indicator and the second indicator produce afluorescence resonance energy transfer signal, and wherein the chemicalreaction of the biopolymer in the presence of the analyte causes thepositions of the first and second indicators to change such that thefluorescence resonance energy transfer signal is altered producing thealteration in the signal.
 236. The method of claim 197, wherein theparticle comprises a polymeric resin, a biopolymer coupled to thepolymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and furthercomprising an indicator coupled to the biopolymer, and wherein thechemical reaction of the biopolymer in the presence of the analytecauses the biopolymer to be cleaved such that a portion of thebiopolymer coupled to the indicator is cleaved from a portion of thebiopolymer coupled to the polymeric resin.
 237. The method of claim 197,wherein the particle comprises a receptor coupled to a polymeric resin,and a probe molecule coupled to the polymeric resin, and wherein theprobe molecule is configured to produce a signal when the receptorinteracts with the analyte during use.
 238. The method of claim 197,wherein the particle comprises a receptor and an indicator coupled to apolymeric resin, wherein the indicator is configured to produce a signalwhen the receptor interacts with the analyte during use.
 239. The methodof claim 197, wherein the particle comprises a receptor and an indicatorcoupled to a polymeric resin, wherein the indicator is configured toproduce a signal when the receptor interacts with the analyte duringuse, and wherein the receptor is coupled to the polymeric resin by afirst linker and wherein the indicator is coupled to the polymeric resinby a second linker.
 240. The method of claim 197, wherein the particlecomprises a receptor and an indicator coupled to a polymeric resin,wherein the indicator is configured to produce a signal when thereceptor interacts with the analyte during use, and wherein the receptoris coupled to the polymeric resin by a first linker and wherein theindicator is coupled to the polymeric resin by a second linker, andwherein the particle further comprises an additional indicator coupledto the polymeric resin by a third linker, wherein the interaction of thereceptor with the analyte causes the indicator and the additionalindicator to interact such that the signal is produced.
 241. The methodof claim 197, wherein the particle comprises a receptor and an indicatorcoupled to a polymeric resin, wherein the indicator is configured toproduce a signal when the receptor interacts with the analyte duringuse, and wherein the receptor is coupled to the polymeric resin by afirst linker and wherein the indicator is coupled to the receptor. 242.The method of claim 197, wherein the particle comprises a receptor andan indicator coupled to a polymeric resin, wherein the indicator isconfigured to produce a signal when the receptor interacts with theanalyte during use, and wherein the receptor is coupled to the polymericresin by a first linker and wherein the indicator is coupled to thereceptor, and wherein the particle further comprises an additionalindicator coupled to the receptor, wherein the interaction of thereceptor with the analyte causes the indicator and the additionalindicator to interact such that the signal is produced.
 243. The methodof claim 197, wherein the particle comprises a receptor and an indicatorcoupled to a polymeric resin, wherein the indicator is configured toproduce a signal when the receptor interacts with the analyte duringuse, and wherein the receptor is coupled to the polymeric resin by afirst linker and wherein the indicator is coupled to the receptor by asecond linker.
 244. The method of claim 197, wherein the particlecomprises a receptor and an indicator coupled to a polymeric resin,wherein the indicator is configured to produce a signal when thereceptor interacts with the analyte during use, and wherein the receptoris coupled to the polymeric resin by a first linker and wherein theindicator is coupled to the receptor by a second linker, and wherein theparticle further comprises an additional indicator coupled to thereceptor, wherein the interaction of the receptor with the analytecauses the indicator and the additional indicator to interact such thatthe signal is produced.
 245. The method of claim 197, wherein theparticle comprises a receptor and an indicator coupled to a polymericresin, wherein the indicator is configured to produce a signal when thereceptor interacts with the analyte during use, and wherein the receptoris coupled to the polymeric resin by a first linker and wherein theindicator is coupled to the first linker.
 246. The method of claim 197,wherein the particle comprises a receptor and an indicator coupled to apolymeric resin, wherein the indicator is configured to produce a signalwhen the receptor interacts with the analyte during use, and wherein thereceptor is coupled to the polymeric resin by a first linker, andwherein the indicator is coupled to the first linker by a second linker.247. The method of claim 197, wherein the particle comprises a receptorand an indicator coupled to a polymeric resin, wherein the indicator isconfigured to produce a signal when the receptor interacts with theanalyte during use, and wherein the receptor is coupled to the polymericresin by a first linker, and wherein the indicator is coupled to thefirst linker by a second linker, and wherein the particle furthercomprises an additional indicator coupled to the receptor, wherein theinteraction of the receptor with the analyte causes the indicator andthe additional indicator to interact such that the signal is produced.248. The method of claim 197, wherein the particle comprises a receptorand an indicator coupled to a polymeric resin, wherein the indicator isconfigured to produce a signal when the receptor interacts with theanalyte during use, and wherein the receptor is coupled to the polymericresin by a first linker, and wherein the indicator is coupled to thefirst linker by a second linker, and wherein the particle furthercomprises an additional indicator coupled to the first linker by a thirdlinker, wherein the interaction of the receptor with the analyte causesthe indicator and the additional indicator to interact such that thesignal is produced.
 249. The method of claim 197, wherein the particlecomprises a receptor and an indicator coupled to a polymeric resin,wherein the indicator is configured to produce a signal when thereceptor interacts with the analyte during use, and wherein theindicator interacts with the receptor in the absence of an analyte. 250.The method of claim 197, wherein the particle comprises a receptor andan indicator coupled to a polymeric resin, wherein the indicator isconfigured to produce a signal when the receptor interacts with theanalyte during use, and wherein the particle further comprises anadditional indicator coupled to the polymeric resin, and wherein theindicator is a first fluorescent dye and wherein the additionalindicator is a second fluorescent dye, and wherein the indicator and theadditional indicator produce a fluorescence resonance energy transfersignal, and wherein the interaction of the analyte with the receptorcauses the distance between the indicator and the additional indicatorto become altered such that the fluorescence resonance energy transfersignal is altered.
 251. The method of claim 197, wherein the particlecomprises a receptor and an indicator coupled to a polymeric resin,wherein the indicator is configured to produce a signal when thereceptor interacts with the analyte during use, and wherein the particlefurther comprises an additional indicator coupled to the polymericresin, wherein the indicator is a fluorescent dye and wherein theadditional indicator is a fluorescence quencher, and wherein theindicator and the additional indicator are positioned such that thefluorescence of the indicator is at least partially quenched by theadditional indicator, and wherein the interaction of the analyte withthe receptor causes the distance between the indicator and theadditional indicator to become altered such that the quenching of thefluorescence of the indicator by the additional indicator is altered.252. The method of claim 197, wherein the particle comprises abiopolymer coupled to a polymeric resin, and wherein the biopolymerundergoes a chemical reaction in the presence of the analyte to producea signal, and wherein the biopolymer undergoes a chemical reaction inthe presence of the analyte such that the signal is altered.
 253. Aparticle for detecting an analyte in a fluid comprising: a polymericresin; a biopolymer coupled to the polymeric resin; and an indicatorsystem coupled to the biopolymer, the indicator system producing asignal, and wherein the biopolymer undergoes a chemical reaction in thepresence of the analyte such that the signal is altered.
 254. Theparticle of claim 253, wherein the particle ranges from about 0.05micron to about 500 microns.
 255. The particle of claim 253, wherein avolume of the particle changes when contacted with the fluid.
 256. Theparticle of claim 253, wherein the chemical reaction comprises cleavageof the biopolymer by the analyte.
 257. The particle of claim 253,wherein the biopolymer comprises a peptide, and wherein the analytecomprises a protease, and wherein the chemical reaction comprisescleavage of the peptide by the protease.
 258. The particle of claim 253,wherein the biopolymer comprises a polynucleotide, and wherein theanalyte comprises a nuclease, and wherein the chemical reactioncomprises cleavage of the polynucleotide by the nuclease.
 259. Theparticle of claim 253, wherein the biopolymer comprises anoligosaccharide, and wherein the analyte comprises an oligosaccharidecleaving agent, and wherein the chemical reaction comprises cleavage ofthe oligosaccharide by the oligosaccharide cleaving agent.
 260. Theparticle of claim 253, wherein the particle indicator system comprises afirst indicator and a second indicator, and wherein the chemicalreaction of the biopolymer in the presence of the analyte causes adistance between the first and second indicators to become altered suchthat the signal is produced.
 261. The particle of claim 253, wherein thefirst indicator is a fluorescent dye and wherein the second indicator isa fluorescent quencher, and wherein the first indicator and the secondindicator are within the Foster energy transfer radius, and wherein thechemical reaction of the biopolymer in the presence of the analytecauses the first and second indicators to move outside the Foster energytransfer radius.
 262. The particle of claim 253, wherein the firstindicator is a fluorescent dye and wherein the second indicator is adifferent fluorescent dye, and wherein the first indicator and thesecond indicator produce a fluorescence resonance energy transfersignal, and wherein the chemical reaction of the biopolymer in thepresence of the analyte causes the positions of the first and secondindicators to change such that the fluorescence resonance energytransfer signal is altered.
 263. The particle of claim 253, wherein theindicator system comprises at least one indicator coupled to thebiopolymer, and wherein the chemical reaction of the biopolymer in thepresence of the analyte causes the biopolymer to be cleaved such that aportion of the biopolymer coupled to the indicator is cleaved from aportion of the biopolymer coupled to the polymeric resin.
 264. Aparticle for detecting an analyte in a fluid comprising: a polymericresin; a receptor coupled to the polymeric resin; and a probe moleculecoupled to the biopolymer, the probe molecule configured to produce asignal when the receptor interacts with the analyte during use.
 265. Theparticle of claim 264, wherein the analyte comprises a metal ion, andwherein the probe molecule produces the signal in response to theinteraction of the metal ion with the receptor.
 266. The particle ofclaim 264, wherein the particles further comprises an additional probemolecule coupled to the polymeric resin, wherein the interaction of thereceptor with the analyte causes the probe molecules to interact suchthat the signal is produced.
 267. The particle of claim 264, wherein thereceptor comprises a polynucleotide.
 268. The particle of claim 264,wherein the receptor comprises a peptide.
 269. The particle of claim264, wherein the receptor comprises an enzyme.
 270. The particle ofclaim 264, wherein the receptor comprises a synthetic receptor.
 271. Theparticle of claim 264, wherein the receptor comprises an unnaturalbiopolymer.
 272. The particle of claim 264, wherein the receptorcomprises an antibody.
 273. The particle of claim 264, wherein thereceptor comprises an antigen.
 274. The particle of claim 264, whereinthe analyte comprises phosphate functional groups, and wherein theparticle is configured to produce the signal in the presence of thephosphate functional groups.
 275. The particle of claim 264, wherein theanalyte comprises bacteria, and wherein the particle is configured toproduce the signal in the presence of the bacteria.
 276. The particle ofclaim 264, wherein the receptor comprises an antibody, an aptamer, anorganic receptor, or an enzyme.
 277. The particle of claim 264, whereinthe probe molecule comprises an indicator, a dye, a quantum particle, ora semi-conductor particle.
 278. A particle for detecting an analyte in afluid comprising: a polymeric resin; a receptor coupled to the polymericresin by a first linker; and an indicator coupled to the first linker,the indicator configured to produce a signal when the receptor interactswith the analyte during use.
 279. The particle of claim 278, wherein thereceptor comprises a polynucleotide.
 280. The particle of claim 278,wherein the receptor comprises a peptide.
 281. The particle of claim278, wherein the receptor comprises a compound of the general formula:(R¹)_(n)—X—(R²)_(m) wherein X comprises carbocyclic systems or C₁-C₁₀alkanes, n is an integer of at least 1, m is an integer of at least 1;and wherein each of R¹ independently represents—(CH₂)_(y)—NR³—C(NR⁴)—NR⁵, —(CH₂)_(y)—NR⁶R⁷, —(CH₂)_(y)—NH—Y,—(CH₂)_(y)—O-Z; where y is an integer of at least 1; where R³, R⁴, andR⁵ independently represent hydrogen, alkyl, aryl, alkyl carbonyl of 1 to10 carbon atoms, or alkoxy carbonyl of 1 to 10 carbon atoms, or R⁴ andR⁵ together represent a cycloalkyl group; where R represents hydrogen,alkyl, aryl, alkyl carbonyl of 1 to 10 carbon atoms, or alkoxy carbonylof 1 to 10 carbon atoms; where R⁷ represents alkyl, aryl, alkyl carbonylof 1 to 10 carbon atoms, or alkoxy carbonyl of 1 to 10 carbon atoms;where R⁶ and R together represent a cycloalkyl group; where Y is apeptide, or hydrogen and where Z is a polynucleotide, an oligosaccharideor hydrogen; and wherein each of R² independently represents hydrogen,alkyl, alkenyl, alkynyl, phenyl, phenylalkyl, arylalkyl, aryl, ortogether with another R² group represent a carbocyclic ring.
 282. Theparticle of claim 278, wherein the receptor comprises an enzyme. 283.The particle of claim 278, wherein the receptor is coupled to the firstlinker by a second linker and wherein the indicator is coupled to thefirst linker by a third linker.
 284. The particle of claim 278, whereinthe receptor is coupled to the first linker by a second linker andwherein the indicator is coupled to the first linker by a third linker,and wherein the indicator interacts with the receptor in the absence ofan analyte.
 285. The particle of claim 278, wherein the particle furthercomprises an additional indicator coupled to the first linker, whereinthe interaction of the receptor with the analyte causes the indicatorand the additional indicator to interact such that the signal isproduced.
 286. The particle of claim 278, wherein the particle furthercomprises an additional indicator coupled to the receptor, wherein theinteraction of the receptor with the analyte causes the indicator andthe additional indicator to interact such that the signal is produced.287. The particle of claim 278, wherein the particle further comprisesan additional indicator coupled to the first linker, and wherein theindicator is a first fluorescent dye and wherein the additionalindicator is a second fluorescent dye, and wherein the indicator and theadditional indicator produce a fluorescence resonance energy transfersignal, and wherein the interaction of the analyte with the receptorcauses the distance between the indicator and the additional indicatorto become altered such that the fluorescence resonance energy transfersignal is altered.
 288. The particle of claim 278, wherein the particlefurther comprises an additional indicator coupled to the first linker,wherein the indicator is a fluorescent dye and wherein the additionalindicator is a fluorescence quencher, and wherein the indicator and theadditional indicator are positioned such that the fluorescence of theindicator is at least partially quenched by the additional indicator,and wherein the interaction of the analyte with the receptor causes thedistance between the indicator and the additional indicator to becomealtered such that the quenching of the fluorescence of the indicator bythe additional indicator is altered.
 289. The particle of claim 278,wherein the particle further comprises an additional indicator coupledto the first linker, wherein the indicator is a fluorescence quencherand wherein the additional indicator is a fluorescent dye, and whereinthe indicator and the additional indicator are positioned such that thefluorescence of the additional indicator is at least partially quenchedby the indicator, and wherein the interaction of the analyte with thereceptor causes the distance between the indicator and the additionalindicator to become altered such that the quenching of the fluorescenceof the additional indicator by the indicator is altered.
 290. Theparticle of claim 278, wherein the particle further comprises anadditional indicator coupled to the receptor, and wherein the indicatoris a first fluorescent dye and wherein the additional indicator is asecond fluorescent dye, and wherein the indicator and the additionalindicator produce a fluorescence resonance energy transfer signal, andwherein the interaction of the analyte with the receptor causes thedistance between the indicator and the additional indicator to becomealtered such that the fluorescence resonance energy transfer signal isaltered.
 291. The particle of claim 278, wherein the particle furthercomprises an additional indicator coupled to the receptor, wherein theindicator is a fluorescent dye and wherein the additional indicator is afluorescence quencher, and wherein the indicator and the additionalindicator are positioned such that the fluorescence of the indicator isat least partially quenched by the additional indicator, and wherein theinteraction of the analyte with the receptor causes the distance betweenthe indicator and the additional indicator to become altered such thatthe quenching of the fluorescence of the indicator by the additionalindicator is altered.
 292. The particle of claim 278, wherein theparticle further comprises an additional indicator coupled to thereceptor, wherein the indicator is a fluorescence quencher and whereinthe additional indicator is a fluorescent dye, and wherein the indicatorand the additional indicator are positioned such that the fluorescenceof the additional indicator is at least partially quenched by theindicator, and wherein the interaction of the analyte with the receptorcauses the distance between the indicator and the additional indicatorto become altered such that the quenching of the fluorescence of theadditional indicator by the indicator is altered.
 293. The particle ofclaim 278, wherein the particle further comprises an additionalindicator coupled to the first linker, wherein the receptor is coupledto the first linker by a second linker, the indicator is coupled to thefirst linker by a third linker and the additional indicator is coupledto the first linker by a fourth linker, and wherein the indicator is afirst fluorescent dye and wherein the additional indicator is a secondfluorescent dye, and wherein the indicator and the additional indicatorproduce a fluorescence resonance energy transfer signal, and wherein theinteraction of the analyte with the receptor causes the distance betweenthe indicator and the additional indicator to become altered such thatthe fluorescence resonance energy transfer signal is altered.
 294. Theparticle of claim 278, wherein the particle further comprises anadditional indicator coupled to the first linker, wherein the receptoris coupled to the first linker by a second linker, the indicator iscoupled to the first linker by a third linker and the additionalindicator is coupled to the first linker by a fourth linker, wherein theindicator is a fluorescent dye and wherein the additional indicator is afluorescence quencher, and wherein the indicator and the additionalindicator are positioned such that the fluorescence of the indicator isat least partially quenched by the additional indicator, and wherein theinteraction of the analyte with the receptor causes the distance betweenthe indicator and the additional indicator to become altered such thatthe quenching of the fluorescence of the indicator by the additionalindicator is altered.
 295. The particle of claim 278, wherein theparticle further comprises an additional indicator coupled to the firstlinker, wherein the receptor is coupled to the first linker by a secondlinker, the indicator is coupled to the first linker by a third linkerand the additional indicator is coupled to the first linker by a fourthlinker, wherein the indicator is a fluorescence quencher and wherein theadditional indicator is a fluorescent dye, and wherein the indicator andthe additional indicator are positioned such that the fluorescence ofthe additional indicator is at least partially quenched by theindicator, and wherein the interaction of the analyte with the receptorcauses the distance between the indicator and the additional indicatorto become altered such that the quenching of the fluorescence of theadditional indicator by the indicator is altered.
 296. The particle ofclaim 278, wherein the particle further comprises an additionalindicator coupled to the receptor, wherein the receptor is coupled tothe first linker by a second linker, the indicator is coupled to thefirst linker by a third linker and the additional indicator is coupledto the receptor by a fourth linker, and wherein the indicator is a firstfluorescent dye and wherein the additional indicator is a secondfluorescent dye, and wherein the indicator and the additional indicatorproduce a fluorescence resonance energy transfer signal, and wherein theinteraction of the analyte with the receptor causes the distance betweenthe indicator and the additional indicator to become altered such thatthe fluorescence resonance energy transfer signal is altered.
 297. Theparticle of claim 278, wherein the particle further comprises anadditional indicator coupled to the receptor, wherein the receptor iscoupled to the first linker by a second linker, the indicator is coupledto the first linker by a third linker and the additional indicator iscoupled to the receptor by a fourth linker, wherein the indicator is afluorescent dye and wherein the additional indicator is a fluorescencequencher, and wherein the indicator and the additional indicator arepositioned such that the fluorescence of the indicator is at leastpartially quenched by the additional indicator, and wherein theinteraction of the analyte with the receptor causes the distance betweenthe indicator and the additional indicator to become altered such thatthe quenching of the fluorescence of the indicator by the additionalindicator is altered.
 298. The particle of claim 278, wherein theparticle further comprises an additional indicator coupled to thereceptor, wherein the receptor is coupled to the first linker by asecond linker, the indicator is coupled to the first linker by a thirdlinker and the additional indicator is coupled to the receptor by afourth linker, wherein the indicator is a fluorescent dye and whereinthe additional indicator is a fluorescence quencher, and wherein theindicator and the additional indicator are positioned such that thefluorescence of the indicator is at least partially quenched by theadditional indicator, and wherein the interaction of the analyte withthe receptor causes the distance between the indicator and theadditional indicator to become altered such that the quenching of thefluorescence of the indicator by the additional indicator is altered.299. The particle of claim 278, wherein the particle further comprisesan additional indicator coupled to the receptor, wherein the receptor iscoupled to the first linker by a second linker, the indicator is coupledto the first linker by a third linker and the additional indicator iscoupled to the receptor by a fourth linker, wherein the indicator is afluorescence quencher and wherein the additional indicator is afluorescent dye, and wherein the indicator and the additional indicatorare positioned such that the fluorescence of the additional indicator isat least partially quenched by the indicator, and wherein theinteraction of the analyte with the receptor causes the distance betweenthe indicator and the additional indicator to become altered such thatthe quenching of the fluorescence of the additional indicator by theindicator is altered.
 300. The particle of claim 278, wherein thepolymeric resin comprises polystyrene-polyethylene glycol-divinylbenzene.
 301. A particle for detecting an analyte in a fluid comprising:a polymeric resin; a biopolymer coupled to the polymeric resin; and anindicator system coupled to the biopolymer, the indicator systemproducing a signal during use, and wherein the biopolymer undergoes achemical reaction in the presence of the analyte such that the signal isaltered during use.
 302. The particle of claim 301, wherein the chemicalreaction comprises cleavage of at least a portion of the biopolymer bythe analyte.
 303. The particle of claim 301, wherein the biopolymercomprises a polynucleotide, and wherein the analyte comprises anuclease, and wherein the chemical reaction comprises cleavage of atleast a portion of the polynucleotide by the nuclease.
 304. The particleof claim 301, wherein the biopolymer comprises an oligosaccharide, andwherein the analyte comprises an oligosaccharide cleaving agent, andwherein the chemical reaction comprises cleavage of at least a portionof the oligosaccharide by the oligosaccharide cleaving agent.
 305. Theparticle of claim 301, wherein the particle indicator system comprises afirst indicator and a second indicator, and wherein the chemicalreaction of the biopolymer in the presence of the analyte causes adistance between the first and second indicators to become altered suchthat the signal is produced.
 306. The particle of claim 689, wherein thefirst indicator is a fluorescent dye and wherein the second indicator isa fluorescence quencher, and wherein the first indicator and the secondindicator are positioned such that the fluorescence of the firstindicator is at least partially quenched by the second indicator, andwherein the chemical reaction of the biopolymer in the presence of theanalyte causes the first and second indicators to move such that thequenching of the fluorescence of the first indicator by the secondindicator is altered.
 307. The particle of claim 689, wherein the firstindicator is a fluorescent dye and wherein the second indicator is adifferent fluorescent dye, and wherein the first indicator and thesecond indicator produce a fluorescence resonance energy transfersignal, and wherein the chemical reaction of the biopolymer in thepresence of the analyte causes the positions of the first and secondindicators to change such that the fluorescence resonance energytransfer signal is altered.
 308. The particle of claim 301, wherein theindicator system comprises at least one indicator coupled to thebiopolymer, and wherein the chemical reaction of the biopolymer in thepresence of the analyte causes the biopolymer to be cleaved such that atleast a portion of the biopolymer coupled to the indicator is cleavedfrom at least a portion of the biopolymer coupled to the polymericresin.
 309. A system for detecting an analyte in a bodily fluidcomprising: a light source; a sensor array, the sensor array comprisinga supporting member comprising at least one cavity formed within thesupporting member; a particle, the particle positioned within thecavity, wherein the particle is configured to produce a signal when theparticle interacts with the analyte in the bodily fluid during use, andwherein the particle comprises a receptor molecule coupled to apolymeric resin; and a detector, the detector being configured to detectthe signal produced by the interaction of the analyte with the particleduring use; wherein the light source and detector are positioned suchthat light passes from the light source, to the particle, and onto thedetector during use.
 310. The system of claim 309, wherein the analytecomprises bacteria, and wherein the receptor molecule comprises anantibody specific for the bacteria in the bodily fluid.
 311. The systemof claim 309, wherein the analyte comprises bacteria, and wherein thereceptor molecule comprises an antigen specific for at least one type ofantibody in the bodily fluid that is produced in response to thebacteria in the bodily fluid.
 312. The system of claim 309, wherein theanalyte comprises mastitis bacteria, and wherein the receptor moleculecomprises an antibody specific for mastitis bacteria.
 313. The system ofclaim 309, wherein the bodily fluid comprises a plurality of bacteria,and wherein the sensor array includes a plurality of cavities with aplurality of particles, wherein each particle comprises a receptorconfigured to produce a signal in the presence of at least one of thebacteria present in the fluid sample.
 314. The system of claim 309,wherein the analyte comprises mycobacterium tuberculosis, and whereinthe receptor molecule comprises an antibody specific for mycobacteriumtuberculosis.
 315. The system of claim 309, wherein the analytecomprises a parasite, and wherein the receptor molecule comprises anantibody specific for the parasite in the bodily fluid.
 316. The systemof claim 309, wherein the analyte comprises a parasite, and wherein thereceptor molecule comprises an antigen specific for at least one type ofantibody in the bodily fluid that is produced in response to theparasite in the bodily fluid.
 317. The system of claim 309, wherein theanalyte comprises heartworm microfilaria, and wherein the receptormolecule comprises an antibody specific for heartworm microfilaria. 318.The system of claim 309, wherein the analyte comprises a virus, andwherein the receptor molecule comprises an antibody specific for thevirus in the bodily fluid.
 319. The system of claim 309, wherein theanalyte comprises a virus, and wherein the receptor molecule comprisesan antigen specific for at least one type of antibody in the bodilyfluid that is produced in response to the virus in the bodily fluid.320. The system of claim 309, wherein the analyte comprises felineleukemia virus, and wherein the receptor molecule comprises an antibodyspecific for feline leukemia virus.
 321. The system of claim 309,wherein the analyte comprises HIV virus, and wherein the receptormolecule comprises an antibody specific for HIV virus.
 322. The systemof claim 309, wherein the analyte comprises hepatitis C virus, andwherein the receptor molecule comprises an antibody specific forhepatitis C virus.
 323. The system of claim 309, wherein the analytecomprises a carbohydrate, and wherein the receptor molecule isconfigured to produce the signal in the presence of a carbohydrate. 324.The system of claim 309, wherein the analyte comprises a steroid, andwherein the receptor molecule is configured to produce the signal in thepresence of a steroid.
 325. The system of claim 309, wherein the analytecomprises a triglyceride, and wherein the receptor molecule isconfigured to produce the signal in the presence of a triglyceride. 326.The system of claim 309, wherein the analyte comprises homocysteine, andwherein the receptor molecule is configured to produce the signal in thepresence of a homocysteine.
 327. The system of claim 309, wherein theanalyte comprises an anticonvulsive drug, and wherein the receptormolecule is configured to produce the signal in the presence of ananticonvulsive drug.
 328. The system of claim 309, wherein the analytecomprises an amphetamine, and wherein the receptor molecule isconfigured to produce the signal in the presence of an amphetamine. 329.The system of claim 309, wherein the analyte comprises a barbiturate,and wherein the receptor molecule is configured to produce the signal inthe presence of an barbiturate.
 330. The system of claim 309, whereinthe analyte comprises a benzodiazepine, and wherein the receptormolecule is configured to produce the signal in the presence of abenzodiazepine.
 331. The system of claim 309, wherein the analytecomprises an opiate, and wherein the receptor molecule is configured toproduce the signal in the presence of an opiate.
 332. The system ofclaim 309, wherein the analyte comprises cocaine, and wherein thereceptor molecule is configured to produce the signal in the presence ofcocaine.
 333. The system of claim 309, wherein the analyte comprisesmarijuana, and wherein the receptor molecule is configured to producethe signal in the presence of marijuana.
 334. The system of claim 309,wherein the analyte comprises an electrolyte, and wherein the receptormolecule is configured to produce the signal in the presence of anelectrolyte.
 335. The system of claim 309, wherein the analyte comprisesfeline leukemia and wherein the receptor comprises feline leukemiaantigen.
 336. The system of claim 309, wherein the light sourcecomprises a light emitting diode.
 337. The system of claim 309, furthercomprising a fluid delivery system coupled to the supporting member.338. The system of claim 309, wherein the detector comprises acharge-coupled device.
 339. The system of claim 309, wherein theparticle ranges from about 0.05 micron to about 500 microns.
 340. Thesystem of claim 309, wherein a volume of the particle changes whencontacted with the fluid.
 341. The system of claim 309, wherein theparticle comprises a receptor molecule coupled to a polymeric resin.342. The system of claim 309, wherein the particle comprises a receptormolecule coupled to a polymeric resin, and wherein the polymeric resincomprises polystyrene-polyethylene glycol-divinyl benzene.
 343. Thesystem of claim 309, wherein the particle comprises a receptor moleculecoupled to a polymeric resin, and wherein the particles furthercomprises a first indicator and a second indicator, the first and secondindicators being coupled to the receptor, wherein the interaction of thereceptor with the analyte causes the first and second indicators tointeract such that the signal is produced.
 344. The system of claim 309,wherein the particle comprises a receptor molecule coupled to apolymeric resin, and wherein the particles further comprises anindicator, wherein the indicator is associated with the receptor suchthat in the presence of the analyte the indicator is displaced from thereceptor to produce the signal.
 345. The system of claim 309, whereinthe supporting member comprises silicon.
 346. The system of claim 309,wherein the system comprises a plurality of particles positioned withina plurality of cavities, and wherein the system is configured tosubstantially simultaneously detect a plurality of analytes in thefluid.
 347. A method of sensing an analyte in a bodily fluid comprising:passing the bodily fluid over a sensor array, the sensor arraycomprising at least one particle positioned within a cavity of asupporting member, wherein the particle is configured to produce asignal when the particle interacts with the analyte in the bodily fluidduring use, and wherein the particle comprises a receptor moleculecoupled to a polymeric resin; and monitoring a spectroscopic change ofthe particle as the fluid is passed over the sensor array, wherein thespectroscopic change is caused by the interaction of the analyte withthe particle.
 348. The method of claim 347, wherein the analytecomprises bacteria, and wherein the receptor molecule comprises anantibody specific for the bacteria in the bodily fluid.
 349. The methodof claim 347, wherein the analyte comprises bacteria, and wherein thereceptor molecule comprises an antigen specific for at least one type ofantibody in the bodily fluid that is produced in response to thebacteria in the bodily fluid.
 350. The method of claim 347, wherein theanalyte comprises mastitis bacteria, and wherein the receptor moleculecomprises an antibody specific for mastitis bacteria.
 351. The method ofclaim 347, wherein the bodily fluid comprises a plurality of bacteria,and wherein the sensor array includes a plurality of cavities with aplurality of particles, wherein each particle comprises a receptorconfigured to produce a signal in the presence of at least one of thebacteria present in the fluid sample.
 352. The method of claim 347,wherein the analyte comprises mycobacterium tuberculosis, and whereinthe receptor molecule comprises an antibody specific for mycobacteriumtuberculosis.
 353. The method of claim 347, wherein the analytecomprises a parasite, and wherein the receptor molecule comprises anantibody specific for the parasite in the bodily fluid.
 354. The methodof claim 347, wherein the analyte comprises a parasite, and wherein thereceptor molecule comprises an antigen specific for at least one type ofantibody in the bodily fluid that is produced in response to theparasite in the bodily fluid.
 355. The method of claim 347, wherein theanalyte comprises heartworm microfilaria, and wherein the receptormolecule comprises an antibody specific for heartworm microfilaria. 356.The method of claim 347, wherein the analyte comprises a virus, andwherein the receptor molecule comprises an antibody specific for thevirus in the bodily fluid.
 357. The method of claim 347, wherein theanalyte comprises a virus, and wherein the receptor molecule comprisesan antigen specific for at least one type of antibody in the bodilyfluid that is produced in response to the virus in the bodily fluid.358. The method of claim 347, wherein the analyte comprises felineleukemia virus, and wherein the receptor molecule comprises an antibodyspecific for feline leukemia virus.
 359. The method of claim 347,wherein the analyte comprises HIV virus, and wherein the receptormolecule comprises an antibody specific for HIV virus.
 360. The methodof claim 347, wherein the analyte comprises hepatitis C virus, andwherein the receptor molecule comprises an antibody specific forhepatitis C virus.
 361. The method of claim 347, wherein the analytecomprises a carbohydrate, and wherein the receptor molecule isconfigured to produce the signal in the presence of a carbohydrate. 362.The method of claim 347, wherein the analyte comprises a steroid, andwherein the receptor molecule is configured to produce the signal in thepresence of a steroid.
 363. The method of claim 347, wherein the analytecomprises a triglyceride, and wherein the receptor molecule isconfigured to produce the signal in the presence of a triglyceride. 364.The method of claim 347, wherein the analyte comprises homocysteine, andwherein the receptor molecule is configured to produce the signal in thepresence of a homocysteine.
 365. The method of claim 347, wherein theanalyte comprises an anticonvulsive drug, and wherein the receptormolecule is configured to produce the signal in the presence of ananticonvulsive drug.
 366. The method of claim 347, wherein the analytecomprises an amphetamine, and wherein the receptor molecule isconfigured to produce the signal in the presence of an amphetamine. 367.The method of claim 347, wherein the analyte comprises a barbiturate,and wherein the receptor molecule is configured to produce the signal inthe presence of an barbiturate.
 368. The method of claim 347, whereinthe analyte comprises a benzodiazepine, and wherein the receptormolecule is configured to produce the signal in the presence of abenzodiazepine.
 369. The method of claim 347, wherein the analytecomprises an opiate, and wherein the receptor molecule is configured toproduce the signal in the presence of an opiate.
 370. The method ofclaim 347, wherein the analyte comprises cocaine, and wherein thereceptor molecule is configured to produce the signal in the presence ofcocaine.
 371. The method of claim 347, wherein the analyte comprisesmarijuana, and wherein the receptor molecule is configured to producethe signal in the presence of marijuana.
 372. The method of claim 347,wherein the analyte comprises an electrolyte, and wherein the receptormolecule is configured to produce the signal in the presence of anelectrolyte.
 373. The method of claim 347, wherein the analyte comprisesfeline leukemia and wherein the receptor comprises feline leukemiaantigen.
 374. The method of claim 347, wherein the spectroscopic changecomprises a change in absorbance of the particle.
 375. The method ofclaim 347, wherein the spectroscopic change comprises a change influorescence of the particle.
 376. The method of claim 347, wherein thespectroscopic change comprises a change in phosphorescence of theparticle.
 377. The method of claim 347, wherein the particle comprises areceptor coupled to a polymeric resin, and further comprising exposingthe particle to an indicator prior to passing the fluid over the sensorarray.
 378. The method of claim 347, wherein the particle comprises areceptor coupled to a polymeric resin, and further comprising exposingthe particle to an indicator prior to passing the fluid over the sensorarray, and wherein a binding strength of the indicator to the receptoris less than a binding strength of the analyte to the receptor.
 379. Themethod of claim 347, wherein the particle comprises a receptor coupledto a polymeric resin, and further comprising exposing the particle to anindicator prior to passing the fluid over the sensor array, and whereinthe indicator is a fluorescent indicator.
 380. The method of claim 347,further comprising treating the fluid with an indicator prior to passingthe fluid over the sensor array, wherein the indicator is configured tocouple with the analyte.
 381. The method of claim 347, wherein theanalyte is bacteria and further comprising breaking down the bacteriaprior to passing the fluid over the sensor array.
 382. The method ofclaim 347, wherein monitoring the spectroscopic change is performed witha CCD device.
 383. The method of claim 347, further comprising measuringthe intensity of the spectroscopic change, and further comprisingcalculating the concentration of the analyte based on the intensity ofthe spectroscopic change.
 384. The method of claim 347, wherein thesupporting member comprises silicon.
 385. The method of claim 347,wherein the spectroscopic change comprises a change in reflectance ofthe particle.
 386. The method of claim 347, wherein the cavity isconfigured such that the fluid entering the cavity passes through thesupporting member.
 387. The method of claim 347, wherein monitoring thespectroscopic change comprises: directing a red light source at theparticle; detecting the absorbance of red light by the particle;directing a green light source at the particle; detecting the absorbanceof green light by the particle; directing a blue light source at theparticle; and detecting the absorbance of blue light by the particle.388. The method of claim 347, wherein the sensor array further comprisesa vacuum chamber coupled to a conduit and the sensor array, and whereinthe chamber is configured to provide a pulling force on the fluid in thesensor array.
 389. The method of claim 347, wherein the fluid is blood.390. The method of claim 347, further comprising passing the fluidthrough a filter prior to passing the fluid over the sensor array. 391.The method of claim 347, further comprising passing the fluid through areagent reservoir prior to passing the fluid over the sensor array. 392.The method of claim 347, wherein the particle comprises a polymericresin, a biopolymer coupled to the polymeric resin, and wherein thebiopolymer undergoes a chemical reaction in the presence of the analyteto produce a signal.
 393. The method of claim 347, wherein the particlecomprises a polymeric resin, a biopolymer coupled to the polymericresin, and wherein the biopolymer undergoes a chemical reaction in thepresence of the analyte to produce a signal, and wherein the signalcomprises an absorbance of the particle.
 394. The method of claim 347,wherein the particle comprises a polymeric resin, a biopolymer coupledto the polymeric resin, and wherein the biopolymer undergoes a chemicalreaction in the presence of the analyte to produce a signal, and whereinthe signal comprises a fluorescence of the particle.
 395. A system fordetecting a nucleic acid analyte in a fluid comprising: a light source;a sensor array, the sensor array comprising a supporting membercomprising at least one cavity formed within the supporting member; aparticle, the particle positioned within the cavity, wherein theparticle is configured to produce a signal when the particle interactswith the nucleic acid analyte during use; a detector, the detector beingconfigured to detect the signal produced by the interaction of thenucleic acid analyte with the particle during use; wherein the lightsource and detector are positioned such that light passes from the lightsource, to the particle, and onto the detector during use.
 396. Thesystem of claim 395, wherein the particle comprises a DNA oligomer andthe nucleic acid analyte comprises DNA, and wherein the DNA oligomer hasa sequence that is complementary to a DNA sequence of the nucleic acidanalyte.
 397. The system of claim 395, wherein the particle comprises anRNA oligomer and the nucleic acid analyte comprises DNA, and wherein theRNA oligomer has a sequence that is complementary to a DNA sequence ofthe nucleic acid analyte.
 398. The system of claim 395, wherein theparticle comprises a DNA oligomer and the nucleic acid analyte comprisesRNA, and wherein the DNA oligomer has a sequence that is complementaryto an RNA sequence of the nucleic acid analyte.
 399. The system of claim395, wherein the particle comprises a RNA oligomer and the nucleic acidanalyte comprises RNA, and wherein the RNA oligomer has a sequence thatis complementary to an RNA sequence of the nucleic acid analyte. 400.The system of claim 395, wherein the particle comprises a DNA-bindingmolecule.
 401. The system of claim 395, wherein the particle comprises aprotein.
 402. The system of claim 395, wherein the particle comprises anenzyme.
 403. The system of claim 395, wherein the particle has asequence that is complementary to the sequence of the nucleic acidanalyte, such that the particle and nucleic acid analyte form a matchedor a mismatched duplex.
 404. The system of claim 403, further comprisingribonuclease, wherein the ribonuclease is configured to dispose of themismatched duplex.
 405. The system of claim 404, wherein theribonuclease is RNase A, RNase T1, or RNase T2.
 406. The system of claim403, further comprising nuclease, wherein the nuclease is configured todispose of the mismatched duplex.
 407. The system of claim 406, whereinthe nuclease is S1 nuclease.
 408. The system of claim 403, furthercomprising a Mispair Recognition Protein, wherein the MispairRecognition Protein is configured to bind the mismatched duplex. 409.The system of claim 408, wherein the Mispair Recognition Proteins isMutS or MutY in combination with thymine glycosylase.
 410. The system ofclaim 403, further comprising a modified form of a mismatch recognitionprotein configured to cleave the mismatched duplex.
 411. The system ofclaim 403, further comprising biotin and avidin, wherein the biotin isconfigured to bind to the mismatched duplex, and wherein the mismatchedduplex bound to biotin is removed by binding to avidin.
 412. The systemof claim 395, further comprising a recombinase protein, wherein theparticle has a sequence that is complementary to the sequence of thenucleic acid analyte, and wherein the recombinase protein catalyzes theformation of a particle analyte duplex.
 413. The system of claim 395,further comprising DNA ligase, wherein: the analyte comprises anoligonucleotide; the particle comprises an oligonucleotide; and the DNAligase is configured to ligate the analyte and the particle, wherein asingle base pair mismatch at a junction of the analyte and particle willprevent ligation.
 414. The system of claim 413, wherein a label iscoupled to the analyte and to the particle.
 415. The system of claim395, wherein the particle comprises an intercalator and a polymericbead.
 416. The system of claim 415, wherein the intercalator ishydroxylamine, potassium permanganate, tetraethyl ammonium acetate,osmium tetroxide, ethidium bromide, POTO, or Texas Red.
 417. The systemof claim 415, further comprising a DNA piperidine, wherein the particlehas a sequence that is complementary to the sequence of the nucleic acidanalyte, such that the particle and nucleic acid analyte form a matchedor a mismatched duplex and wherein the DNA piperidine is configured tocleave the mismatched duplex.
 418. The system of claim 395, wherein thelight source comprises a light emitting diode.
 419. The system of claim395, further comprising a fluid delivery system coupled to thesupporting member.
 420. The system of claim 395, wherein the detectorcomprises a charge-coupled device.
 421. The system of claim 395, whereinthe particle ranges from about 0.05 micron to about 500 microns. 422.The system of claim 395, wherein a volume of the particle changes whencontacted with the fluid.
 423. The system of claim 395, wherein theparticle comprises a receptor molecule coupled to a polymeric resin.424. The system of claim 423, wherein the polymeric resin comprisespolystyrene-polyethylene glycol-divinyl benzene.
 425. The system ofclaim 423, wherein the particles further comprises a first indicator anda second indicator, the first and second indicators being coupled to thereceptor, wherein the interaction of the receptor with the analytecauses the first and second indicators to interact such that the signalis produced.
 426. The system of claim 423, wherein the particles furthercomprises an indicator, wherein the indicator is associated with thereceptor such that in the presence of the analyte the indicator isdisplaced from the receptor to produce the signal.
 427. The system ofclaim 395, wherein the supporting member comprises silicon.
 428. Asensor array for detecting a nucleic acid analyte in a fluid comprising:a supporting member, wherein a plurality of cavities are formed withinthe supporting member; a plurality of particles, at least one particlebeing positioned in each of the cavities, wherein the particles areconfigured to produce a signal when the particles interact with theanalyte.
 429. The system of claim 429, and wherein the system isconfigured to substantially simultaneously detect a plurality ofanalytes in the fluid.
 430. The sensor array of claim 429, wherein theparticles comprise a plurality of intercalculators.
 431. The sensorarray of claim 429, further comprising channels in the supportingmember, wherein the channels are configured to allow the fluid to flowthrough the channels into and away from the cavity.
 432. The sensorarray of claim 429, further comprising a barrier layer positioned overthe cavity, the barrier layer being configured to inhibit dislodgment ofthe particle during use.
 433. The sensor array of claim 432, wherein thebarrier layer comprises a substantially transparent cover platepositioned over the cavity, and wherein the cover plate is positioned afixed distance over the cavity such that the fluid can enter the cavity.434. The sensor array of claim 429, wherein the supporting membercomprises a plastic material.
 435. The sensor array of claim 429,wherein the supporting member comprises a dry film photoresist material.436. The sensor array of claim 429, wherein the cavities are configuredsuch that the fluid entering the cavities passes through the supportingmember during use.
 437. The sensor array of claim 436, furthercomprising a pump coupled to the supporting member, wherein the pump isconfigured to direct the fluid towards the cavities.
 438. The sensorarray of claim 437, wherein a channel is formed in the supportingmember, and wherein the channel couples the pump to the cavity such thatthe fluid flows through the channel to the cavity during use.
 439. Thesensor array of claim 437, further comprising a vacuum apparatus,wherein the vacuum apparatus is coupled to the cavity, and wherein thevacuum apparatus is configured to pull the fluid through the cavityduring use.
 440. A method of sensing a nucleic acid analyte in a fluidcomprising: passing a first fluid over a sensor array, the sensor arraycomprising at least one particle positioned within a cavity of asupporting member, the particle comprising a receptor coupled to apolymeric resin, wherein the receptor is configured to produce a signalwhen the receptor interacts with the nucleic acid analyte during use;and monitoring a signal produced by the particle as the fluid is passedover the sensor array, wherein the presence of the signal indicates thepresence of the analyte.
 441. The method of claim 440, wherein thesignal comprises a change in the absorbance of the particle.
 442. Themethod of claim 440, wherein the signal comprises a change in thefluorescence of the particle.
 443. The method of claim 440, wherein thereceptor comprises RNA, and wherein the analyte comprises DNA, andfurther comprising treating the particle with a ribonuclease afterpassing the fluid over the sensor array.
 444. The method of claim 440,wherein the receptor comprises RNA, and wherein the analyte comprisesRNA, and further comprising treating the particle with a ribonucleaseafter passing the fluid over the sensor array.
 445. The method of claim440, wherein the receptor comprises DNA, and wherein the analytecomprises DNA, and further comprising treating the particle with an S1nuclease after passing the fluid over the sensor array.
 446. The methodof claim 440, wherein the receptor comprises RNA, and wherein theanalyte comprises RNA, and further comprising treating the particle withan S1 nuclease after passing the fluid over the sensor array.
 447. Themethod of claim 440, wherein the receptor comprises DNA, and wherein theanalyte comprises DNA, and further comprising treating the particle withan intercalator and piperidine after passing the fluid over the sensorarray.
 448. The method of claim 440, wherein the receptor comprises aprotein, and wherein the analyte comprises DNA.
 449. The method of claim440, wherein the receptor comprises a protein, and wherein the analytecomprises RNA.
 450. The method of claim 440, wherein the receptorcomprises DNA, and wherein the analyte comprises DNA, and furthercomprising treating the particle with DNA ligase after passing the fluidover the sensor array.
 451. The method of claim 440, further comprisingamplifying the nucleic acid present in the fluid using a polymerasechain reaction prior to passing the fluid over the sensor array. 452.The method of claim 440, further comprising amplifying the nucleic acidpresent in the fluid using a ligase chain reaction prior to passing thefluid over the sensor array.
 453. The method of claim 440, where in theanalyte comprises DNA, and further comprising forming an RNA copy of theanalyte prior to passing the fluid over the sensor array.
 454. Themethod of claim 440, wherein the receptor further comprises anindicator.
 455. The method of claim 440, further comprising passing asecond fluid over the sensor array, the second fluid comprising anintercalator.
 456. The method of claim 455, wherein the intercalator ishydroxylamine, potassium permanganate, tetraethyl ammonium acetate,osmium tetroxide, ethidium bromide, POTO, or Texas Red.
 457. The methodof claim 455, further comprising passing DNA piperidine over the sensorarray.
 458. The method of claim 440, wherein further comprising passinga second fluid over the sensor array, the second fluid comprisingMispair Recognition Proteins.
 459. The method of claim 440, wherein thereceptor comprises an oligonucleotide and wherein the analyte comprisesan oligonucleotide, and further comprising passing DNA ligase over thesensor array and scoring ligation by assaying for labels on theoligoneucleotides becoming present in a single molecule.